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Multiple mutations have been seen to undergo convergent evolution in SARS-CoV-2 variants of concern. One such evolution occurs in Beta, Gamma, and Omicron variants at three amino acid positions K417, E484, and N501 in the receptor binding domain of the spike protein. We examined the physical mechanisms underlying the convergent evolution of three mutations K417T/E484K/N501Y by delineating the individual and collective effects of mutations on binding to angiotensin converting enzyme 2 receptor, immune escape from neutralizing antibodies, protein stability, and expression. Our results show that each mutation serves a distinct function that improves virus fitness supporting its positive selection, even though individual mutations have deleterious effects that make them prone to negative selection. Compared to the wild-type, K417T escapes Class 1 antibodies and has increased stability and expression; however, it has decreased receptor binding. E484K escapes Class 2 antibodies; however, it has decreased receptor binding, stability, and expression. N501Y increases receptor binding; however, it has decreased stability and expression. When these mutations come together, the deleterious effects are mitigated due to the presence of compensatory effects. Triple mutant K417T/E484K/N501Y has increased receptor binding, escapes both Class 1 and Class 2 antibodies, and has similar stability and expression as that of the wild-type. These results show that the convergent evolution of multiple mutations enhances viral fitness on different fronts by balancing both positive and negative selection and improves the chances of selection of mutations together.

Human Pumilio (hPUM) is a structurally well-analyzed RNA-binding protein that has been used recently for artificial RNA binding. Structural analysis revealed that amino acids at positions 12, 13, and 16 in the repeats from R1 to R8 each contact one specific RNA base in the eight-nucleotide RNA target. The functions of the N- and C-terminal flanking repeats R1' and R8', however, remain unclear. Here, we report how the repeats contribute to overall RNA binding. We first prepared three mutants in which R1' and/or R8' were deleted and then analyzed RNA binding using gel shift assays. The assays showed that all deletion mutants bound to their target less than the original hPUM, but that R1' contributed more than R8', unlike Drosophila PUM. We next investigated which amino acid residues of R1' or R8' were responsible for RNA binding. With detailed analysis of the protein tertiary structure, we found a hydrophobic core in each of the repeats. We therefore mutated all hydrophobic amino residues in each core to alanine. The gel shift assays with the resulting mutants revealed that both hydrophobic cores contributed to the RNA binding: especially the hydrophobic core of R1' had a significant influence. In the present study, we demonstrated that the flanking R1' and R8' repeats are indispensable for RNA binding of hPUM and suggest that hydrophobic R1'-R1 interactions may stabilize the whole hPUM structure.

The geminivirus replication protein, Rep, has long been recognized as a high-value target for control of geminivirus infections as this protein is highly conserved and essential for viral replication and proliferation. In addition, inhibition of viral replication has been pursued through various antiviral strategies with varying degrees of success, including inhibitory peptides that target Rep. While much effort has centered around sequence characterization of the Rep protein and inhibitory peptides, detailed structural analysis has been missing. This study computationally investigated the presence of common structural features within these inhibitory peptides and if these features could inform if a particular peptide will bind Rep and/or interfere with viral replication. Molecular dynamics simulations of the inhibitory peptide library showed that simply possessing stable structural features does not inform interference of viral replication regardless of the binding of Rep. Additionally, nearly all known Rep inhibitory peptides sample a conserved β-sheet structural motif, possibly informing structure-function relationships in binding Rep. In particular, two peptides (A22 and A64) characterized by this structural motif were computationally docked against a wide variety of geminivirus Rep proteins to determine a mechanism of action. Computational docking revealed these peptides utilize a common Rep protein sequence motif for binding, HHN-x 1/2 -Q. The results identified residues in both Rep and the inhibitory peptides that play a significant role in the interaction, establishing the foundation for a rational structure-based design approach for the construction of both broadly reactive and geminivirus species-specific inhibitors.

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity among the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against S protein from infectious virus, cultured in Vero cells. We find patterns that are conserved across all samples, and this can be associated with site-specific stalling of glycan maturation that acts as a highly sensitive reporter of protein structure. Molecular dynamics simulations of a fully glycosylated spike support a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

Active DNA transposases like the Drosophila P element transposase (DmTNP) undergo oligomerization as a prerequisite for transposition. Human THAP9 (hTHAP9) is a catalytically active but functionally uncharacterized homologue of DmTNP. Here we report (using co-immunoprecipitation, pull down, colocalization, and proximity ligation assays) that both full length and truncated hTHAP9 (corresponding to amino-terminal DNA binding and predicted coiled coil domains) undergo homo-oligomerization, predominantly in the nuclei of HEK293T cells. Interestingly, the oligomerization is shown to be partially mediated by DNA. However, mutating the leucines (either individually or together) or deleting the predicted coiled coil region did not significantly affect oligomerization. Thus, we highlight the importance of DNA and the amino-terminal regions of hTHAP9 for their ability to form higher-order oligomeric states. We also report that Hcf-1, THAP1, THAP10, and THAP11 are possible protein interaction partners of hTHAP9. Elucidating the functional relevance of the different putative oligomeric state(s) of hTHAP9 would help answer questions about its interaction partners as well as its unknown physiological roles.

The NF-κB family of transcription factors is a key regulator of the immune response in the vertebrates. The family comprises five proteins that function as dimers formed in various combinations among the members, with the RelA-p50 dimer being physiologically the most abundant. While most of the 15 possible dimers are scarcely present in the cell with some remaining experimentally undetected to date, there are specific gene sets that are only activated by certain sparsely populated NF-κB dimers. The mechanism of transcription activation of such specific genes that are activated only by specific NF-κB dimers remains unclear. Here we show that the dimer interfacial residues control the stabilization of the global hydrogen bond network of the NF-κB dimerization domain, which, in turn, controls the thermodynamic stabilization of different NF-κB dimers. The relatively low thermodynamic stability of the RelA-RelA homodimer is critical as it facilitates the formation of the more stable RelA-p50 heterodimer. Through the modulation of the thermodynamic stability of the RelA-RelA homodimer, the kinetics of the RelA-p50 heterodimer formation can be regulated. This phenomenon provides an insight into the mechanism of RelA-RelA specific target gene regulation in physiology.

ZntA from Escherichia coli confers resistance to toxic concentrations of Pb 2+ , Zn 2+ , and Cd 2+ . It is a member of the P 1B -ATPase transporter superfamily, which includes the human Cu + -transporting proteins ATP7A and ATP7B. P 1B -type ATPases typically have a hydrophilic N-terminal metal-binding domain and eight transmembrane helices. A splice variant of ATP7B was reported, which has 100-fold higher night-specific expression in the pineal gland; it lacks the entire N-terminal domain and the first four transmembrane helices. Here, we report our findings with Δ231-ZntA, a similar truncation we created in ZntA. Δ231-ZntA has no in vivo and greatly reduced in vitro activity. It binds one metal ion per dimer at the transmembrane site, with a 15-19000-fold higher binding affinity, indicating highly significant changes in the dimer structure of Δ231-ZntA relative to that of ZntA. Cd 2+ has the highest affinity for Δ231-ZntA, in contrast to ZntA, which has the highest affinity for Pb 2+ . Site-specific mutagenesis of the metal-binding residues, 392 Cys, 394 Cys, and 714 Asp, showed that there is considerable flexibility at the metal-binding site, with any two of these three residues able to bind Zn 2+ and Pb 2+ unlike in ZntA. However, Cd 2+ binds to only 392 Cys and 714 Asp, with 394 Cys not involved in Cd 2+ binding. Three-dimensional homology models show that there is a dramatic difference between the ZntA and Δ231-ZntA dimer structures, which help to explain these observations. Therefore, the first four transmembrane helices in ZntA and P 1B -type ATPases play an important role in maintaining the correct dimer structure.

Neisseria gonorrhoeae possesses a programmed recombination system that allows the bacteria to alter the major subunit of the type IV pilus, pilin or PilE. An alternate DNA structure known as a guanine quadruplex (G4) is required for pilin antigenic variation (pilin Av). The G-C base pairs within the G4 motif are required for pilin Av, but simple mutation of the loop bases does not affect pilin Av. We show that more substantial changes to the loops, in both size and nucleotide composition, with the core guanines unchanged, decrease or abrogate pilin Av. We investigated why these loop changes might influence the efficiency of pilin Av. RecA is a recombinase required for pilin Av that can bind the pilE G4 in vitro. RecA binds different G4 structures with altered loops with varied affinities. However, changes in RecA binding affinities to the loop mutants do not absolutely correlate with the pilin Av phenotypes. Interestingly, the yeast RecA ortholog, Rad51, also binds the pilE G4 structure with a higher affinity than it binds single-stranded DNA, suggesting that RecA G4 binding is conserved in eukaryotic orthologs. The thermal stability the pilE G4 structure and its loop mutants showed that the parental G4 structure had the highest melting temperature, and the melting temperature of the loop mutants correlated with pilin Av phenotype. These results suggest that the folding kinetics and stability of G4 structures are important for the efficiency of pilin Av.

The modular biosynthetic pathway of ribosomally synthesized and post-translationally modified peptides (RiPPs) enhances their engineering potential for exploring new structures and biological functions. The ω-ester-containing peptides (OEPs), a subfamily of RiPPs, have distinct side-to-side ester or amide linkages and frequently present more than one macrocyclic domain in a "beads-on-a-string" structure. In an effort to improve the engineering potential of RiPPs, we present here the idea that the multidomain architecture of an OEP, plesiocin, can be exploited to create a bifunctional modified peptide. Characterization of plesiocin variants revealed that strong chymotrypsin inhibition relies on the bicyclic structure of the domain in which a leucine residue in the hairpin loop functions as a specificity determinant. Four domains of plesiocin promote simultaneous binding of multiple enzymes, where the C-terminal domain binds chymotrypsin most efficiently. Using this information, we successfully engineered a plesiocin variant in which two different domains inhibit chymotrypsin and trypsin. This result suggests that the multidomain architecture of OEPs is a useful platform for engineering multifunctional hybrid RiPPs.

Despite decades of research on ion-sensing proteins, gaps persist in the understanding of ion binding affinity and selectivity even in well-studied proteins such as calmodulin. Site-directed mutagenesis is a powerful and popular tool for addressing outstanding questions about biological ion binding and is employed to selectively deactivate binding sites and insert chromophores at advantageous positions within ion binding structures. However, even apparently nonperturbative mutations can distort the binding dynamics they are employed to measure. We use Fourier transform infrared (FTIR) and ultrafast two-dimensional infrared (2D IR) spectroscopy of the carboxylate asymmetric stretching mode in calmodulin as a mutation- and label-independent probe of the conformational perturbations induced in calmodulin's binding sites by two classes of mutation, tryptophan insertion and carboxylate side-chain deletion, commonly used to study ion binding in proteins. Our results show that these mutations not only affect ion binding but also induce changes in calmodulin's conformational landscape along coordinates not probed by vibrational spectroscopy, remaining invisible without additional perturbation of binding site structure. Comparison of FTIR line shapes with 2D IR diagonal slices provides a clear example of how nonlinear spectroscopy produces well-resolved line shapes, refining otherwise featureless spectral envelopes into more informative vibrational spectra of proteins.

Promoters are the key drivers of gene expression and are largely responsible for the regulation of cellular responses to time and environment. In Escherichia coli, decades of studies have revealed most, if not all, of the sequence elements necessary to encode promoter function. Despite our knowledge of these motifs, it is still not possible to predict the strength and regulation of a promoter from primary sequence alone. Here we develop a novel multiplexed assay to study promoter function in E. coli by building a site-specific genomic recombination-mediated cassette exchange system that allows for the facile construction and testing of large libraries of genetic designs integrated into precise genomic locations. We build and test a library of 10898 σ70 promoter variants consisting of all combinations of a set of eight -35 elements, eight -10 elements, three UP elements, eight spacers, and eight backgrounds. We find that the -35 and -10 sequence elements can explain approximately 74% of the variance in promoter strength within our data set using a simple log-linear statistical model. Simple neural network models explain >95% of the variance in our data set by capturing nonlinear interactions with the spacer, background, and UP elements.

Chemokines play important roles in immune defense by directing migration of leukocytes and serve as key promoters of tumorigenesis and metastasis. This study explores the molecular mechanisms of recognition and activation of two homologous chemokine receptors, CXCR1 and CXCR2, using CXCL8 analogues with residue substitutions in the conserved Glu4Leu5Arg6 (ELR) triad. Analysis of the binding of CXCL8 analogues to CXCR1 is consistent with the two-site model for signal recognition of CXCR1, whereas analysis of the binding of CXCL8 analogues to CXCR2 supported a single-site model for signal recognition of CXCR2. The CXCL8-Arg6His analogue stimulated calcium release, phosphorylation of ERK1/2, and chemotaxis in cells expressing CXCR1. However, CXCL8-Arg6His failed to stimulate calcium release and chemotaxis in cells expressing CXCR2, although it stimulated phosphorylation of ERK1/2, indicating that CXCL8-Arg6His operated as a classical CXCR2 biased agonist. The CXCL8-Glu4AlaLeu5AlaArg6His analogue was inactive in cells expressing CXCR1 and CXCR2. These findings suggest that the Glu4Leu5 motif in CXCL8 is essential for activation of CXCR1 and CXCR2. Importantly, CXCL8-Glu4AlaLeu5AlaArg6His blocked specifically the calcium release and chemotaxis of cells expressing CXCR1 but not of cells expressing CXCR2. CXCL8-Glu4AlaLeu5AlaArg6His was identified as the first specific CXCR1 antagonist. The binding of CXCL8-ELR6H to CXCR1 created a Zn 2+ coordination site at the receptor activation domain responsible for calcium release, as ZnCl 2 specifically blocked CXCL8-Arg6His-induced calcium release without affecting CXCL8-induced calcium release. This work provides the basis for further exploration of the activation mechanisms of chemokine receptors and will assist in the design of the next generation of modulators of CXCR1 and CXCR2.

The intrinsically disordered transactivation domains of HIF-1α and CITED2 compete for binding of the TAZ1 domain of the CREB-binding protein by a unidirectional allosteric mechanism involving direct competition for shared binding sites, ternary complex formation, and TAZ1 conformational changes. To gain insight into the mechanism by which CITED2 displaces HIF-1α from TAZ1, we used nuclear magnetic resonance spin relaxation methods to obtain an atomic-level description of the picosecond to nanosecond backbone dynamics that contribute to TAZ1 binding and competition. We show that HIF-1α and CITED2 adopt different dynamics in their complexes with TAZ1, with flexibility observed for HIF-1α in regions that would maintain accessibility for CITED2 to bind to TAZ1 and facilitate subsequent HIF-1α dissociation. In contrast, critical regions of CITED2 adopt a rigid structure in its complex with TAZ1, minimizing the ability of HIF-1α to compete for binding. We also find that TAZ1, previously thought to be a rigid scaffold for binding of disordered protein ligands, displays altered backbone dynamics in its various bound states. TAZ1 is more rigid in its CITED2-bound state than in its free state or in complex with HIF-1α, with increased rigidity observed not only in the CITED2 binding site but also in regions of TAZ1 that undergo conformational changes between the HIF-1α- and CITED2-bound structures. Taken together, these data suggest that backbone dynamics in TAZ1, as well as in the HIF-1α and CITED2 ligands, play a role in modulating the occupancy of TAZ1 and highlight the importance of characterizing both binding partners in molecular interactions.

To be fully active and participate in the metabolism of phosphorylated nucleotides, most nucleoside diphosphate kinases (NDPKs) have to assemble into stable hexamers. Here we studied the role played by six intersubunit salt bridges R80-D93 in the stability of NDPK from the pathogen Mycobacterium tuberculosis ( Mt). Mutating R80 into Ala or Asn abolished the salt bridges. Unexpectedly, compensatory stabilizing mechanisms appeared for R80A and R80N mutants and we studied them by biochemical and structural methods. The R80A mutant crystallized into space group I222 that is unusual for NDPK, and its hexameric structure revealed the occurrence at the trimer interface of a stabilizing hydrophobic patch around the mutation. Functionally relevant, a trimer of the R80A hexamer showed a remodeling of the binding site. In this conformation, the cleft of the active site is more open, and then active His117 is more accessible to substrates. H/D exchange mass spectrometry analysis of the wild type and the R80A and R80N mutants showed that the remodeled region of the protein is highly solvent accessible, indicating that equilibrium between open and closed conformations is possible. We propose that such equilibrium occurs in vivo and explains how bulky substrates access the catalytic His117.

A 29-residue peptide (MP01), identified by in vitro selection for reactivity with a small molecule perfluoroaromatic, was modified and characterized using experimental and computational techniques, with the goal of understanding the molecular basis of its reactivity. These studies identified a six-amino acid point mutant (MP01-Gen4) that exhibited a reaction rate constant of 25.8 ± 1.8 M -1 s -1 at pH 7.4 and room temperature, approximately 2 orders of magnitude greater than that of its progenitor sequence and 3 orders of magnitude greater than background cysteine reactivity. MP01-Gen4 appeared to be conformationally dynamic and exhibited several properties reminiscent of larger protein molecules, including denaturant-sensitive structure and reactivity. We believe the majority of the reaction rate enhancement can be attributed to interaction of MP01-Gen4 with the perfluoroaromatic probe, which was found to stabilize a helical conformation of both MP01-Gen4 and nonreactive Cys-to-Ser or Cys-to-Ala variants. These findings demonstrate the ability of dynamic peptides to access proteinlike reaction mechanisms and the potential of perfluoroaromatic functionality to stabilize small peptide folds.

The fibrillation of misfolded neurodegenerative disease-related proteins has been extensively studied over the past few decades, but the initial oligomerization has been rarely examined even though some recent evidence indicated that small protein oligomers are more neurotoxic than long protein fibers. It is rather difficult to study the initial oligomerization (nucleation) because most experimental methods, such as the β-sheet-related fluorescence assay and X-ray diffraction, are unable to detect small structureless protein oligomers. In this study, we have successfully developed a method to link a short thermally sensitive poly(N-isopropylacrylamide) (PNIPAM) chain to a model protein, Sup35NM, at a specific 31st residue site (Sup35NM-31m-PNIPAM) via the efficient thiol-ene Michael addition reaction. The oligomerization was studied by a combination of laser light scattering, the thioflavin T assay, and transmission electron microscopy. We found that the lag phase of Sup35NM was delayed from 12 to >24 h under the physiological condition after the PNIPAM linkage. The oligomerization and fibrillation constants decreased from 5.0 × 10 -3 to 1.5 × 10 -3 h -1 and from 3.0 × 10 -2 to 1.8 × 10 -2 μM -1 h -1 , respectively, presumably because of the steric hindrance introduced by the PNIPAM chain. Moreover, after initiating the oligomerization, we found that the oligomer distribution in the first 6 h repeatedly and quantitatively follows the Smoluchowski coagulation model. Our study paves the way for controllably and quantitatively studying the oligomerization kinetics of amyloidogenic proteins. In addition, by investigating the effects of different small molecules on the oligomerization kinetics, we should be able to screen potential drugs to slow the development of neurodegenerative diseases.

The binding affinity of the human papillomavirus type 6 E2 protein is strongly mediated by the sequence of the DNA linker region, with high affinity for the AATT linker and low affinity for the CCGG linker. When two terminal leucine residues are removed from the protein, the level of binding to both strands increases, but unequally, resulting in a significant decrease in selectivity for the AATT linker strand. To rationalize this behavior, we performed molecular dynamics simulations of the wild-type and mutant protein in the apo state and bound to DNA with high-affinity AATT and low-affinity CCGG linker strands. While no stable contacts were made between the β2-β3 loop and DNA in the wild type, this loop was repositioned in the mutant complexes and formed electrostatic contacts with the DNA backbone. More contacts were formed when the mutant was bound to the CCGG linker strand than to the AATT linker strand, resulting in a more favorable change in interaction energy for the CCGG strand. In addition, significant differences in correlated motions were found, which further explained the differences in binding. The simulations suggest that β2-β3 loop motions are responsible for the increased affinity and decreased selectivity of the mutant protein.

Alzheimer's disease (AD) and Parkinson's disease (PD) are caused by β-amyloid (Aβ) and α-synuclein (αS), respectively. Ample evidence suggests that these two pathogenic proteins are closely linked and have a synergistic effect on eliciting neurodegenerative disorders. However, the pathophysiological consequences of Aβ and αS coexistence are still elusive. Here, we show that large-sized αS oligomers, which are normally difficult to form, are readily generated by Aβ42-seeding and that these oligomers efficiently hamper neuronal SNARE-mediated vesicle fusion. The direct binding of the Aβ-seeded αS oligomers to the N-terminal domain of synaptobrevin-2, a vesicular SNARE protein, is responsible for the inhibition of fusion. In contrast, large-sized Aβ42 oligomers (or aggregates) or the products of αS incubated without Aβ42 have no effect on vesicle fusion. These results are confirmed by examining PC12 cell exocytosis. Our results suggest that Aβ and αS cooperate to escalate the production of toxic oligomers, whose main toxicity is the inhibition of vesicle fusion and consequently prompts synaptic dysfunction.

A recombinant carboxylesterase, cloned from Pseudomonas putida and designated as rPPE, is capable of catalyzing the bioresolution of racemic 2-acetoxy-2-(2'-chlorophenyl)acetate (rac-AcO-CPA) with excellent (S)-enantioselectivity. Semirational design of the enzyme showed that the W187H variant could increase the activity by ∼100-fold compared to the wild type (WT) enzyme. In this study, we performed all-atom molecular dynamics (MD) simulations of both apo-rPPE and rPPE in complex with (S)-AcO-CPA to gain insights into the origin of the increased catalysis in the W187H mutant. Our results show differential binding of (S)-AcO-CPA in the WT and W187H enzymes, especially the interactions of the substrate with the two active site residues Ser159 and His286. The replacement of Trp187 by His leads to considerable structural rearrangement in the active site of W187H. Unlike in the WT rPPE, the cap domain in the W187 mutant shows an open conformation in the simulations of both apo and substrate-bound enzymes. This open conformation exposes the catalytic triad to the solvent through a water accessible channel, which may facilitate the entry of the substrate and/or the exit of the product. Binding free energy calculations confirmed that the substrate binds more strongly in W187H than in WT. On the basis of these computational results, we further predicted that the mutations W187Y and D287G might also be able to increase the substrate binding and thus improve the enzyme's catalytic efficiency. Experimental binding and kinetic assays on W187Y and D287G show improved catalytic efficiency over WT, but not W187H. Contrary to our prediction, W187Y shows slightly decreased substrate binding coupled with a 100-fold increase in turnover rate, while in D287G the substrate binding is 8 times stronger but with a slightly reduced turnover rate. Our work provides important molecular-level insights into the binding of the (S)-AcO-CPA substrate to carboxylesterase rPPEs, which will help guide future development of more efficient rPPE variants.

The spike (S) protein of severe acute respiratory syndrome-associated CoV (SARS-CoV) mediates membrane fusion and viral entry. These events involve structural rearrangements, including heteromerization between two heptad repeats (HR1 and HR2) to form a trimer of dimers as a six-helix bundle (6-HB), a quaternary protein structure that brings two distant clusters of hydrophobic sequences into the proximity of each other, the internal fusion peptide (IFP) preceding HR1, and the highly conserved tryptophan (Trp)-rich membrane proximal external region (MPER) following HR2. Here, we show that MPER can undergo self-oligomerization and heteromerization with IFP, events that are Trp-dependent. To delineate the roles of Trp residues of MPER in forming these quaternary structures and interacting with membranes, we employed a panel of synthetic peptides: MPER peptide (M-wt) and its alanine (Ala) and phenylalanine (Phe) analogues. Ala substitutions of Trp inhibited its association with cellular membranes. Chemical cross-linking experiments showed that M-wt can self-interact to form oligomers and cross-interact with IFP23, a synthetic IFP peptide, to form a heterohexamer. In comparison, little high-order oligomer was formed between M-wt and fusion peptide. The specific interaction between M-wt and IFP23 was confirmed by immunofluorescence staining experiments. In aqueous solutions, both M-wt and IFP23 displayed random secondary structures that became helical in hydrophobic solvents. Triple-Ala substitutions of Trp in M-wt, but not the corresponding triple-Phe analogue, disrupted oligomerization of M-wt and hetero-oligomerization of M-wt with IFP23. Overall, our results show that Trp residues of MPER play a key role in maintaining the structure and functions of MPER, allowing it to interact with IFP to form a MPER-IFP heteromer, a putative quaternary structure extending from the 6-HB, and function in membrane fusion. Finally, we showed that a MPER peptide could serve as an inhibitor in the entry process.

Activation of the protein tyrosine kinase receptors requires the coupling of ligand binding to a change in both the proximity and orientation of the single transmembrane (TM) helices of receptor monomers to allow transphosphorylation of the receptor kinase domain. We make use of peptides corresponding to the TM and juxtamembrane (JM) regions of the fibroblast growth factor receptor 3 to assess how mutations in the TM region (G380R and A391E), which lead to receptor activation, influence the orientation of the TM domain and interactions of the intracellular JM sequence with the membrane surface. On the basis of fluorescence and Fourier transform infrared spectroscopy, we find that both activating mutations change the TM helix tilt angle relative to the membrane normal and release the JM region from the membrane. These results suggest a general mechanism regarding how the TM-JM region functionally bridges the extracellular and intracellular regions for these receptors.

The design of new optogenetic tools for controlling protein function would be facilitated by the development of protein scaffolds that undergo large, well-defined structural changes upon exposure to light. Domain swapping, a process in which a structural element of a monomeric protein is replaced by the same element of another copy of the same protein, leads to a well-defined change in protein structure. We observe domain swapping in a variant of the blue light photoreceptor photoactive yellow protein in which a surface loop is replaced by a well-characterized protein-protein interaction motif, the E-helix. In the domain-swapped dimer, the E-helix sequence specifically binds a partner K-helix sequence, whereas in the monomeric form of the protein, the E-helix sequence is unable to fold into a binding-competent conformation and no interaction with the K-helix is seen. Blue light irradiation decreases the extent of domain swapping (from Kd = 10 μM to Kd = 300 μM) and dramatically enhances the rate, from weeks to <1 min. Blue light-induced domain swapping thus provides a novel mechanism for controlling of protein-protein interactions in which light alters both the stability and the kinetic accessibility of binding-competent states.

Asparagine (N)-linked glycosylation is essential for efficient protein folding in the endoplasmic reticulum (ER) and anterograde trafficking through the secretory pathway. N-Glycans are attached to nascent polypeptides at consensus sites, N-X-T/S (X ≠ P), by one of two enzymatic isoforms of the oligosaccharyltransferase (OST), STT3A or STT3B. Here, we examined the effect of the consensus site X and hydroxyl residue on the distributions of co- and post-translational N-glycosylation of a type I transmembrane glycopeptide scaffold. Using rapid radioactive pulse-chase experiments to resolve co-translational (STT3A) and post-translational (STT3B) events, we determined that NXS consensus sites containing large hydrophobic and negatively charged middle residues are frequently skipped by STT3A during protein translation. Post-translational modification of the cotranslationally skipped sites by STT3B was similarly hindered by the middle X residue, resulting in hypoglycosylation of NXS sites containing large hydrophobic and negatively charged side chains. In contrast, NXT consensus sites (barring NWT) were efficiently modified by the cotranslational machinery, reducing STT3B's role in modifying consensus sites skipped during protein translation. A strong correlation between cotranslational N-glycosylation efficiency and the rate of post-translational N-glycosylation was determined, showing that the OST STT3A and STT3B isoforms are similarly influenced by the hydroxyl and middle X consensus site residues. Substituting various middle X residues into an OST eubacterial homologous structure revealed that small and polar consensus site X residues fit well in the peptide binding site whereas large hydrophobic and negatively charged residues were harder to accommodate, indicating conserved enzymatic mechanisms for the mammalian OST isoforms.

Interleukin-1 receptor-associated kinase 1 (IRAK1) is a crucial signaling kinase in the immune system, involved in Toll-like receptor signaling. Vasodilator-stimulated phosphoprotein (VASP) is a central player in cell migration that regulates actin polymerization and connects signaling events to cytoskeletal remodeling. A VASP–IRAK1 interaction is thought to be important in controlling macrophage migration in response to protein kinase C-ε activation. We show that the monomeric VASP EVH1 domain directly binds to the 168WPPPP172 motif in the IRAK1 undefined domain (IRAK1-UD) with moderate affinity (KDApp = 203 ± 3 μM). We further show that this motif adopts distinct cis and trans isomers for the Trp168–Pro169 peptide bond with nearly equal populations, and that binding to the VASP EVH1 domain is specific for the trans isomer, coupling binding to isomerization. Nuclear magnetic resonance line shape analysis and tryptophan fluorescence experiments reveal the complete kinetics and thermodynamics of the binding reaction, showing diffusion-limited binding to the trans isomer followed by slow, isomerization-dependent binding. We further demonstrate that the peptidyl-prolyl isomerase cyclophilin A (CypA) catalyzes isomerization of the Trp168–Pro169 peptide bond and accelerates binding of the IRAK1-UD to the VASP EVH1 domain. We propose that binding of IRAK1 to tetrameric VASP is regulated by avidity through the assembly of IRAK1 onto receptor-anchored signaling complexes and that an isomerase such as CypA may modulate IRAK1 signaling in vivo. These studies demonstrate a direct interaction between IRAK1 and VASP and suggest a potential mechanism for how this interaction might be regulated by both assembly of IRAK1 onto an activated signaling complex and PPIase enzymes.

In this study, we demonstrate that U1A-RNA molecular recognition is mediated by a combined mechanism of conformational selection and induced fit. The binding of U1A to RNA has been discussed in the context of induced fit that involves the reorientation of the α-helix in the C-terminal region (Helix-C) of U1A to permit RNA access only when U1A correctly recognizes RNA. However, according to our molecular dynamics simulations, even in the absence of RNA, Helix-C spontaneously reoriented to permit RNA access. Nonetheless, such a conformational change was still incomplete. Helix-C was often partially or even fully unfolded and in an infrequent RNA-accessible conformation, which can be detected using state-of-the-art nuclear magnetic resonance methodology. These results suggest that the formation of an energetically stabilized complex is promoted by specific interactions between U1A and RNA. In conclusion, in the recognition of RNA by U1A protein, we propose a combined mechanism that requires the reorientation of Helix-C and the subsequent contact with RNA through conformational selection, although the stabilization of the U1A-RNA complex is caused by induced fit. We further propose a modification to the conventional assumption regarding the mechanism of U1A-RNA molecular recognition.

Single-stranded DNA (ssDNA)-binding protein (SSB) protects ssDNA from degradation and recruits other proteins for DNA replication and repair. Escherichia coli SSB is the prototypical eubacterial SSB in a family of tetrameric SSBs. It consists of a structurally well-defined ssDNA binding domain (OB-domain) and a disordered C-terminal domain (C-domain). The eight-residue C-terminal segment of SSB (C-peptide) mediates the binding of SSB to many different SSB-binding proteins. Previously published nuclear magnetic resonance (NMR) data of the monomeric state at pH 3.4 showed that the C-peptide binds to the OB-domain at a site that overlaps with the ssDNA binding site, but investigating the protein at neutral pH is difficult because of the high molecular mass and limited solubility of the tetramer. Here we show that the C-domain is highly mobile in the SSB tetramer at neutral pH and that binding of the C-peptide to the OB-domain is so weak that most of the C-peptides are unbound even in the absence of ssDNA. We address the problem of determining intramolecular binding affinities in the situation of fast exchange between two states, one of which cannot be observed by NMR and cannot be fully populated. The results were confirmed by electron paramagnetic resonance spectroscopy and microscale thermophoresis. The C-peptide-OB-domain interaction is shown to be driven primarily by electrostatic interactions, so that binding of 1 equiv of (dT)35 releases practically all C-peptides from the OB-domain tetramer. The interaction is much more sensitive to NaCl than to potassium glutamate, which is the usual osmolyte in E. coli. As the C-peptide is predominantly in the unbound state irrespective of the presence of ssDNA, long-range electrostatic effects from the C-peptide may contribute more to regulating the activity of SSB than any engagement of the C-peptide by the OB-domain.

Catalytic subunits of the IκB kinase (IKK), IKK1/IKKα, and IKK2/IKKβ function in vivo as dimers in association with the necessary scaffolding subunit NEMO/IKKγ. Recent X-ray crystal structures of IKK2 suggested that dimerization might be mediated by a smaller protein-protein interaction than previously thought. Here, we report that removal of a portion of the scaffold dimerization domain (SDD) of human IKK2 yields a kinase subunit that remains monomeric in solution. Expression in baculovirus-infected Sf9 insect cells and purification of this engineered monomeric human IKK2 enzyme allows for in vitro analysis of its substrate specificity and mechanism of activation. We find that the monomeric enzyme, which contains all of the amino-terminal kinase and ubiquitin-like domains as well as the more proximal portions of the SDD, functions in vitro to direct phosphorylation exclusively to residues S32 and S36 of its IκBα substrate. Thus, the NF-κB-inducing potential of IKK2 is preserved in the engineered monomer. Furthermore, we observe that our engineered IKK2 monomer readily autophosphorylates activation loop serines 177 and 181 in trans. However, when residues that were previously observed to interfere with IKK2 trans autophosphorylation in transfected cells are mutated within the context of the monomer, the resulting Sf9 cell expressed and purified proteins were significantly impaired in their trans autophosphorylation activity in vitro. This study further defines the determinants of substrate specificity and provides additional evidence in support of a model in which activation via trans autophosphorylation of activation loop serines in IKK2 requires transient assembly of higher-order oligomers.

The toxin RelE is a ribosome-dependent endoribonuclease implicated in diverse cellular processes, including persistence. During amino acid starvation, RelE inhibits translation by cleaving ribosomal A-site mRNA. Although RelE is structurally similar to other microbial endoribonucleases, the active-site amino acid composition differs substantially and lacks obvious candidates for general acid-base functionality. Highly conserved RelE residues (Lys52, Lys54, Arg61, Arg81, and Tyr87) surround the mRNA scissile phosphate, and specific 16S rRNA contacts further contribute to substrate positioning. We used a single-turnover kinetic assay to evaluate the catalytic importance of individual residues in the RelE active site. Within the context of the ribosome, RelE rapidly cleaves A-site mRNA at a rate similar to those of traditional ribonucleases. Single-turnover rate constants decreased between 10(2)- and 10(6)-fold for the RelE active-site mutants of Lys52, Lys54, Arg61, and Arg81. RelE may principally promote catalysis via transition-state charge stabilization and leaving-group protonation, in addition to achieving in-line substrate positioning in cooperation with the ribosome. This kinetic analysis complements structural information to provide a foundation for understanding the molecular mechanism of this atypical endoribonuclease.

Tryptophan is hydrophobic, bulky, and the rarest amino acid found in nutrients. Accordingly, the import machinery can be specialized evolutionarily. Our previous study in Saccharomyces cerevisiae demonstrated that tryptophan import by the high-affinity tryptophan permease Tat2 is accompanied by a large volume increase during substrate import. Nevertheless, the mechanisms by which the permease mediates tryptophan recognition and permeation remain to be elucidated. Here we determined amino acid residues essential for Tat2-mediated tryptophan import. By means of random mutagenesis in combination with site-directed mutagenesis based on crystallographic studies of the Escherichia coli arginine/agmatine antiporter AdiC, we identified 15 amino acid residues in the Tat2 transmembrane domains (TMDs) 1, -3, -5, -8, and -10, which are responsible for tryptophan uptake. T98, Y167, and E286 were assumed to form the central cavity in Tat2. G97/T98 and E286 were located within the putative α-helix break in TMD1 and TMD6, respectively, which are highly conserved among yeast amino acid permeases and bacterial solute transporters. Given the conformational change in AdiC upon substrate binding, G97/T98 and E286 of Tat2 were assumed to mediate a structural shift from an outward-open to a tryptophan-bound-occluded structure upon tryptophan binding, and T320, V322, and F324 became stabilized in TMD7. Such dynamic structural changes may account for the large volume increase associated with tryptophan import occurring concomitantly with a movement of water molecules from the tryptophan binding site. We also propose the working hypothesis that E286 mediates the proton influx that is coupled to tryptophan import.

Dss1 is an intrinsically unstructured polypeptide that partners with the much larger Brh2 protein, the BRCA2 ortholog in Ustilago maydis, to form a tight complex. Mutants lacking Dss1 have essentially the same phenotype as mutants defective in Brh2, implying that through physical interaction Dss1 serves as a positive activator of Brh2. Dss1 associates with Brh2 through an interaction surface in the carboxy-terminal region. Certain derivatives of Brh2 lacking this interaction surface remain highly competent in DNA repair as long as a DNA-binding domain is present. However, the Dss1-independent activity raises the question of what function might be met in the native protein by having Brh2 under Dss1 control. Using a set of Brh2 fusions and truncated derivatives, we show here that Dss1 is capable of exerting control when there is a cognate Dss1-interacting surface present. We find that association of Dss1 attenuates the DNA binding potential of Brh2 and that the amino-terminal domain of Brh2 helps evict Dss1 from its carboxy-terminal interaction surface. The findings presented here add to the notion that Dss1 serves in a regulatory capacity to dictate order in association of Brh2's amino-terminal and carboxy-terminal domains with DNA.

The replication of human immunodeficiency virus type 1 (HIV-1) can be profoundly inhibited by the natural ligands of two major HIV-1 coreceptors, CXCR4 and CCR5. Stromal cell-derived factor-1α (SDF-1α) is a natural ligand of CXCR4. We have recently developed a synthetic biology approach of using synthetically and modularly modified (SMM)-chemokines to dissect various aspects of the structure-function relationship of chemokines and their receptors. Here, we used this approach to design novel SMM-SDF-1α analogues containing unnatural N-methylated residues in the amino terminus to investigate whether the polypeptide main chain amide bonds in the N-terminus of SDF-1α play a role in SDF-1α signaling via CXCR4 and/or receptor internalization. The results show that SDF-1α analogues with a modified N-methylated main chain at position 2, 3, or 5 retain significant CXCR4 binding and yet completely lose signaling activities. Furthermore, a representative N-methylated analogue has been shown to be incapable of causing CXCR4 internalization. These results suggest that the ability of SDF-1α to activate CXCR4 signaling and internalization is dependent upon the main chain amide bonds in the N-terminus of SDF-1α. This study demonstrates the feasibility and value of applying a synthetic biology approach to chemically engineer natural proteins and peptide ligands as probes of important biological functions that are not addressed by other biological techniques.

N-Terminal methylation of free α-amino groups is a post-translational modification of proteins that was first described 30 years ago but has been studied very little. In this modification, the initiating M residue is cleaved and the exposed α-amino group is mono-, di-, or trimethylated by NRMT, a recently identified N-terminal methyltransferase. Currently, all known eukaryotic α-amino-methylated proteins have a unique N-terminal motif, M-X-P-K, where X is A, P, or S. NRMT can also methylate artificial substrates in vitro in which X is G, F, Y, C, M, K, R, N, Q, or H. Methylation efficiencies of N-terminal amino acids are variable with respect to the identity of X. Here we use in vitro peptide methylation assays and substrate immunoprecipitations to show that the canonical M-X-P-K methylation motif is not the only one recognized by NRMT. We predict that N-terminal methylation is a widespread post-translational modification and that there is interplay between N-terminal acetylation and N-terminal methylation. We also use isothermal calorimetry experiments to demonstrate that NRMT can efficiently recognize and bind to its fully methylated products.

The family 4 uracil-DNA glycosylase from the hyperthermophilic organism Archaeoglobus fulgidus (AFUDG) is responsible for the removal of uracil in DNA as the first step in the base excision repair (BER) pathway. AFUDG contains a large solvent-exposed peptide region containing an α helix and loop anchored on each end via ligation of two cysteine thiolates to a [4Fe-4S](2+) cluster. We propose that this region plays a similar role in DNA damage recognition as a smaller iron-sulfur cluster loop (FCL) motif in the structurally unrelated BER glycosylases MutY and Endonuclease III and therefore refer to this region as the "pseudo-FCL" in AFUDG. In order to evaluate the importance of this region, three positively charged residues (Arg 86, Arg 91, Lys 100) and the anchoring Cys residues (Cys 85, Cys 101) within this motif were replaced with alanine, and the effects of these replacements on uracil excision in single- and double-stranded DNA were evaluated. These results show that this region participates and allows for efficient recognition and excision of uracil within DNA. Notably, R86A AFUDG exhibited reduced activity for uracil removal only within double-stranded DNA, suggesting an importance in duplex disruption and extrusion of the base as part of the excision process. In addition, mutation of the [4Fe-4S](2+) cluster cysteine ligands at the ends of the pseudo-FCL to alanine reduced the uracil excision efficiency, suggesting the importance of anchoring the loop via coordination to the cluster. In contrast, K100A AFUDG exhibited enhanced uracil excision activity, providing evidence for the importance of the loop conformation and flexibility. Taken together, the results herein provide evidence that the pseudo-FCL motif is involved in DNA binding and catalysis, particularly in duplex DNA contexts. This work underscores the requirement of an ensemble of interactions, both distant and in proximity to the damaged site, for accurate and efficient uracil excision.

Transfer messenger RNA (tmRNA) and small binding protein B (SmpB) are the main components of the trans-translation rescue machinery that releases stalled ribosomes from defective mRNAs. Little is known about how SmpB binding affects the conformation of the tRNA-like domain (TLD) of tmRNA. It has been previously hypothesized that the absence of a D stem in the TLD provides flexibility in the elbow region of tmRNA, which can be stabilized by its interaction with SmpB. Here, we have used Förster resonance energy transfer to characterize the global structure of the tRNA-like domain of tmRNA in the presence and absence of SmpB and as a function of Mg(2+) concentration. Our results show tight and specific binding of SmpB to tmRNA. Surprisingly, our data show that the global conformation and flexibility of tmRNA do not change upon SmpB binding. However, Mg(2+) ions induce an 11 Å compaction in the tmRNA structure, suggesting that the flexibility in the H2a stem may allow different conformations of tmRNA as the TLD and mRNA-like domain need to be positioned differently while moving through the ribosome.

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes. Knowledge of how this is regulated is important. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic. To investigate the dynamic properties of CaM-NOS interactions, we determined the solution structure of CaM bound to the inducible NOS (iNOS) and endothelial NOS (eNOS) CaM binding region peptides. In addition, we investigated the effect of CaM phosphorylation. Tyrosine 99 (Y99) of CaM is reported to be phosphorylated in vivo. We have produced a phosphomimetic Y99E CaM to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. All three mammalian NOS isoforms were included in the investigation. Our results show that a phosphomimetic Y99E CaM significantly reduces the maximal synthase activity of eNOS by 40% while having little effect on nNOS or iNOS activity. A comparative nuclear magnetic resonance study between phosphomimetic Y99E CaM and wild-type CaM bound to the eNOS CaM binding region peptide was performed. This investigation provides important insights into how the increased electronegativity of a phosphorylated CaM protein affects the binding, dynamics, and activation of the NOS enzymes.

Human small heat shock protein 27 (Hsp27) undergoes concentration-dependent equilibrium dissociation from an ensemble of large oligomers to a dimer. This phenomenon plays a critical role in Hsp27 chaperone activity in vitro enabling high affinity binding to destabilized proteins. In vivo dissociation, which is regulated by phosphorylation, controls Hsp27 role in signaling pathways. In this study, we explore the sequence determinants of Hsp27 dissociation and define the structural basis underlying the increased affinity of Hsp27 dimers to client proteins. A systematic cysteine mutagenesis is carried out to identify residues in the N-terminal domain important for the equilibrium between Hsp27 oligomers and dimers. In addition, spin-labels were attached to the cysteine mutants to enable electron paramagnetic resonance (EPR) analysis of residue environment and solvent accessibility in the context of the large oligomers, upon dissociation to the dimer, and following complex formation with the model substrate T4 Lysozyme (T4L). The mutagenic analysis identifies residues that modulate the equilibrium dissociation in favor of the dimer. EPR analysis reveals that oligomer dissociation disrupts subunit contacts leading to the exposure of Hsp27 N-terminal domain to the aqueous solvent. Moreover, regions of this domain are highly dynamic with no evidence of a packed core. Interaction between T4L and sequences in this domain is inferred from transition of spin-labels to a buried environment in the substrate/Hsp27 complex. Together, the data provide the first structural analysis of sHSP dissociation and support a model of chaperone activity wherein unstructured and highly flexible regions in the N-terminal domain are critical for substrate binding.

Bone marrow stromal cell antigen 2 (BST-2) inhibits the release of enveloped viruses from the cell surface. Various viral counter measures have been discovered, which allow viruses to escape BST-2 restriction. Human immunodeficiency virus type 1 (HIV-1) encodes viral protein U (Vpu) that interacts with BST-2 through their transmembrane domains and causes the downregulation of cell surface BST-2. In this study, we used a computer modeling method to establish a molecular model to investigate the binding interface of the transmembrane domains of BST-2 and Vpu. The model predicts that the interface is composed of Vpu residues I6, A10, A14, A18, V25, and W22 and BST-2 residues L23, I26, V30, I34, V35, L41, I42, and T45. Introduction of mutations that have been previously reported to disrupt the Vpu-BST-2 interaction led to a calculated higher binding free energy (MMGBSA), which supports our molecular model. A pharmacophore was also generated on the basis of this model. Our results provide a precise model that predicts the detailed interaction occurring between the transmembrane domains of Vpu and BST-2 and should facilitate the design of anti-HIV agents that are able to disrupt this interaction.

ANTXR1 is a type I membrane protein that binds the protective antigen (PA) component of anthrax toxin. The cytosolic domain of ANTXR1 has a novel actin-binding region that influences the interaction of the ectodomain with PA. Here, we have investigated features of the cytosolic domain of ANTXR1 that reduce the association of the receptor with PA. We mutated a stretch of conserved acidic amino acids adjacent to the actin-binding region and found that the mutation increased the affinity for monomeric actin in vitro. ANTXR1 bearing this mutation exhibited increased association with the cytoskeleton and bound less PA compared to the wild-type receptor, confirming the inverse correlation between the two interactions. To determine whether binding of actin is sufficient to regulate the ectodomain, we replaced the actin-binding region of ANTXR1 with that from the yeast protein abp140 and with the WH2 domain of WAVE2. Although both of these domains bound monomeric actin in vitro, only the sequence from abp140 reduced binding of PA to a hybrid receptor. The actin binding regions of ANTXR1 and abp140, but not the WH2 domain, colocalized with actin stress fibers, which suggested that filamentous actin regulates ANTXR1. Consistent with this notion, disruption of actin filaments using latrunculin A increased the amount of PA bound to cells. This work provides evidence that cytoskeletal dynamics regulate ANTXR1 function.

The enzyme As(III) S-adenosylmethionine methyltransferase (EC 2.1.1.137) (ArsM or AS3MT) is found in members of every kingdom, from bacteria to humans. In these enzymes, there are three conserved cysteine residues at positions 72, 174, and 224 in the CmArsM orthologue from the thermophilic eukaryotic alga Cyanidioschyzon sp. 5508. Substitution of any of the three led to loss of As(III) methylation. In contrast, a C72A mutant still methylated trivalent methylarsenite [MAs(III)]. Protein fluorescence of a single-tryptophan mutant reported binding of As(III) or MAs(III). As(GS)(3) and MAs(GS)(2) bound significantly faster than As(III), suggesting that the glutathionylated arsenicals are preferred substrates for the enzyme. Protein fluorescence also reported binding of Sb(III), and the purified enzyme methylated and volatilized Sb(III). The results suggest that all three cysteine residues are necessary for the first step in the reaction, As(III) methylation, but that only Cys174 and Cys224 are required for the second step, methylation of MAs(III) to dimethylarsenite [DMAs(III)]. The rate-limiting step was identified as the conversion of DMAs(III) to trimethylarsine, and DMAs(III) accumulates as the principal product.

Phosphomannomutase/phosphoglucomutase contributes to the infectivity of Pseudomonas aeruginosa, retains and reorients its intermediate by 180°, and rotates domain 4 to close the deep catalytic cleft. Nuclear magnetic resonance (NMR) spectra of the backbone of wild-type and S108C-inactivated enzymes were assigned to at least 90%. (13)C secondary chemical shifts report excellent agreement of solution and crystallographic structure over the 14 α-helices, C-capping motifs, and 20 of the 22 β-strands. Major and minor NMR peaks implicate substates affecting 28% of assigned residues. These can be attributed to the phosphorylation state and possibly to conformational interconversions. The S108C substitution of the phosphoryl donor and acceptor slowed transformation of the glucose 1-phosphate substrate by impairing k(cat). Addition of the glucose 1,6-bisphosphate intermediate accelerated this reaction by 2-3 orders of magnitude, somewhat bypassing the defect and apparently relieving substrate inhibition. The S108C mutation perturbs the NMR spectra and electron density map around the catalytic cleft while preserving the secondary structure in solution. Diminished peak heights and faster (15)N relaxation suggest line broadening and millisecond fluctuations within four loops that can contact phosphosugars. (15)N NMR relaxation and peak heights suggest that domain 4 reorients slightly faster in solution than domains 1-3, and with a different principal axis of diffusion. This adds to the crystallographic evidence of domain 4 rotations in the enzyme, which were previously suggested to couple to reorientation of the intermediate, substrate binding, and product release.

Steroid receptors define a family of ligand-activated transcription factors. Recent work has demonstrated that the receptors regulate distinct but overlapping gene networks, yet the mechanisms by which they do so remain unclear. We previously determined the microscopic binding energetics for progesterone receptor (PR) isoform assembly at promoters containing multiple response elements. We found that the two isoforms (PR-A and PR-B) share nearly identical dimerization and intrinsic DNA binding free energies but maintain large differences in cooperative free energy. Moreover, cooperativity can be modulated by monovalent ion binding and promoter layout, suggesting that differences in cooperativity might control isoform-specific promoter occupancy and thus receptor function. To determine whether cooperative binding energetics are common to other members of the steroid receptor family, we dissected the thermodynamics of estrogen receptor-α (ER-α):promoter interactions. We find that the ER-α intrinsic DNA binding free energy is identical to that of the PR isoforms. This was expected, noting that receptor DNA binding domains are highly conserved. Unexpectedly, ER-α generates negligible cooperativity-orders of magnitude less than predicted based on our studies of the PR isoforms. However, analysis of the cooperativity term suggests that it reflects a balance between highly favorable cooperative stabilization and unfavorable promoter bending. Moreover, ER-α cooperative free energy is compensated for by a large increase in dimerization free energy. Collectively, the results demonstrate that steroid receptors differentially partition not only cooperative energetics but also dimerization energetics. We speculate that this ability serves as a framework for regulating receptor-specific promoter occupancy and thus receptor-specific gene regulation.

Factor (F) VIII functions as a cofactor in FXase, markedly accelerating the rate of FIXa-catalyzed activation of FX. Earlier work identified a FX-binding site having μM affinity within the COOH-terminal region of the FVIIIa A1 subunit. In the present study, surface plasmon resonance (SPR), ELISA-based binding assays, and chemical cross-linking were employed to assess an interaction between FX and the FVIII light chain (A3C1C2 domains). SPR and ELISA-based assays showed that FVIII LC bound to immobilized FX (K(d) = 165 and 370 nM, respectively). Furthermore, active site-modified activated protein C (DEGR-APC) effectively competed with FX in binding FVIII LC (apparent K(i) = 82.7 nM). Western blotting revealed that the APC-catalyzed cleavage rate at Arg(336) was inhibited by FX in a concentration-dependent manner. A synthetic peptide comprising FVIII residues 2007-2016 representing a portion of an APC-binding site blocked the interaction of FX and FVIII LC (apparent K(i) = 152 μM) and directly bound to FX (K(d) = 7.7 μM) as judged by SPR and chemical cross-linking. Ala-scanning mutagenesis of this sequence revealed that the A3C1C2 subunit derived from FVIII variants Thr2012Ala and Phe2014Ala showed 1.5- and 1.8-fold increases in K(d) for FX, whereas this value using the A3C1C2 subunit from a Thr2012Ala/Leu2013Ala/Phe2014Ala triple mutant was increased >4-fold. FXase formed using this LC triple mutant demonstrated an ~4-fold increase in the K(m) for FX. These results identify a relatively high affinity and functional FX site within the FVIIIa A3C1C2 subunit and show a contribution of residues Thr2012 and Phe2014 to this interaction.

The crystal structure of the unliganded form of Bacillus stearothermophilus phosphofructokinase (BsPFK) was determined using molecular replacement to 2.8 Å resolution (Protein Data Bank entry 3U39 ). The apo BsPFK structure serves as the basis for the interpretation of any structural changes seen in the binary or ternary complexes. When the apo BsPFK structure is compared with the previously published liganded structures of BsPFK, the structural impact that the binding of the ligands produces is revealed. This comparison shows that the apo form of BsPFK resembles the substrate-bound form of BsPFK, a finding that differs from previous predictions.

The KsgA methyltransferase is universally conserved and plays a key role in regulating ribosome biogenesis. KsgA has a complex reaction mechanism, transferring a total of four methyl groups onto two separate adenosine residues, A1518 and A1519, in the small subunit rRNA. This means that the active site pocket must accept both adenosine and N(6)-methyladenosine as substrates to catalyze formation of the final product N(6),N(6)-dimethyladenosine. KsgA is related to DNA adenosine methyltransferases, which transfer only a single methyl group to their target adenosine residue. We demonstrate that part of the discrimination between mono- and dimethyltransferase activity lies in a single residue in the active site, L114; this residue is part of a conserved motif, known as motif IV, which is common to a large group of S-adenosyl-L-methionine-dependent methyltransferases. Mutation of the leucine to a proline mimics the sequence found in DNA methyltransferases. The L114P mutant of KsgA shows diminished overall activity, and its ability to methylate the N(6)-methyladenosine intermediate to produce N(6),N(6)-dimethyladenosine is impaired; this is in contrast to a second active site mutation, N113A, which diminishes activity to a level comparable to L114P without affecting the methylation of N(6)-methyladenosine. We discuss the implications of this work for understanding the mechanism of KsgA's multiple catalytic steps.

The penultimate reaction in the oxidative degradation of nicotinate (vitamin B(3)) to fumarate in several species of aerobic bacteria is the hydrolytic deamination of maleamate to maleate, catalyzed by maleamate amidohydrolase (NicF). Although it has been considered a model system for bacterial degradation of N-heterocyclic compounds, only recently have gene clusters that encode the enzymes of this catabolic pathway been identified to allow detailed investigations concerning the structural basis of their mechanisms. Here, the Bb1774 gene from Bordetella bronchiseptica RB50, putatively annotated as nicF, has been cloned, and the recombinant enzyme, overexpressed and purified from Escherichia coli, is shown to catalyze efficiently the hydrolysis of maleamate to maleate and ammonium ion. Steady-state kinetic analysis of the reaction by isothermal titration calorimetry (ITC) established k(cat) and K(M) values (pH 7.5 and 25 °C) of 11.7 ± 0.2 s(-1) and 128 ± 6 μM, respectively. The observed K(D) of the NicF·maleate (E·P) complex, also measured by ITC, is approximated to be 3.8 ± 0.4 mM. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The structure provides insight concerning the roles of potential catalytically important residues, most notably a conserved catalytic triad (Asp29, Lys117, and Cys150) observed in the proximity of a conserved non-proline cis-peptide bond within a small cavity that is likely the active site. On the basis of this structural information, the hydrolysis of maleamate is proposed to proceed by a nucleophilic addition-elimination sequence involving the thiolate side chain of Cys150.

Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC. (19)F NMR experiments showed that binding of the inhibitor 5-fluoro-1H-pyrimidin-2-one (5FPy) to the wild-type yCD causes an upfield shift, indicating that the bound inhibitor is in the hydrated form, mimicking the transition state or the tetrahedral intermediate in the activation of 5FC. However, binding of 5FPy to the E64A mutant enzyme causes a downfield shift, indicating that the bound 5FPy remains in an unhydrated form in the complex with the mutant enzyme. (1)H and (15)N NMR analysis revealed trans-hydrogen bond D/H isotope effects on the hydrogen of the amide of Glu64, indicating that the carboxylate of Glu64 forms two hydrogen bonds with the hydrated 5FPy. ONIOM calculations showed that the wild-type yCD complex with the hydrated form of the inhibitor 1H-pyrimidin-2-one is more stable than the initial binding complex, and in contrast, with the E64A mutant enzyme, the hydrated inhibitor is no longer favored and the conversion has a higher activation energy, as well. The hydrated inhibitor is stabilized in the wild-type yCD by two hydrogen bonds between it and the carboxylate of Glu64 as revealed by (1)H and (15)N NMR analysis. To explore the functional role of Glu64 in catalysis, we investigated the deamination of cytosine catalyzed by the E64A mutant by ONIOM calculations. The results showed that without the assistance of Glu64, both proton transfers before and after the formation of the tetrahedral reaction intermediate become partially rate-limiting steps. The results of the experimental and computational studies together indicate that Glu64 plays a critical role in both the binding and the chemical transformation in the conversion of the prodrug 5FC to the anticancer drug 5-fluorouracil.

DNA polymerase delta (Pol δ) is a central enzyme for eukaryotic DNA replication and repair. Pol δ is a complex of four subunits p125, p68, p50, and p12. The functional properties of Pol δ are largely determined by its interaction with its DNA sliding clamp PCNA (proliferating cellular nuclear antigen). The regulatory mechanisms that govern the association of Pol δ with PCNA are largely unknown. In this study, we identified S458, located in the PCNA-interacting protein (PIP-Box) motif of p68, as a phosphorylation site for PKA. Phosphomimetic mutation of S458 resulted in a decrease in p68 affinity for PCNA as well as the processivity of Pol δ. Our results suggest a role of phosphorylation of the PIP-motif of p68 as a molecular switch that dynamically regulates the functional properties of Pol δ.

The zinc finger protein EVI1 is causally associated with acute myeloid leukemogenesis, and inhibition of its function with a small molecule therapeutic may provide effective therapy for EVI1-expressing leukemias. In this paper we describe the development of a pyrrole-imidazole polyamide to specifically block EVI1 binding to DNA. We first identify essential domains for leukemogenesis through structure-function studies on both EVI1 and the t(3;21)(q26;q22)-derived RUNX1-MDS1-EVI1 (RME) protein, which revealed that DNA binding to the cognate motif GACAAGATA via the first of two zinc finger domains (ZF1, encompassing fingers 1-7) is essential transforming activity. To inhibit DNA binding via ZF1, we synthesized a pyrrole-imidazole polyamide 1, designed to bind to a subsite within the GACAAGATA motif and thereby block EVI1 binding. DNase I footprinting and electromobility shift assays revealed a specific and high affinity interaction between polyamide 1 and the GACAAGATA motif. In an in vivo CAT reporter assay using NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as an EVI1-responsive reporter, polyamide 1 completely blocked EVI1-responsive reporter activity. Growth of a leukemic cell line bearing overexpressed EVI1 was also inhibited by treatment with polyamide 1, while a control cell line lacking EVI1 was not. Finally, colony formation by RME was attenuated by polyamide 1 in a serial replating assay. These studies provide evidence that a cell permeable small molecule may effectively block the activity of a leukemogenic transcription factor and provide a valuable tool to dissect critical functions of EVI1 in leukemogenesis.

RNA is an important biological target because it plays essential roles in many pathogenic and normal cellular processes. The design of inhibitors that target RNA involves optimization of noncovalent interactions, including van der Waals, hydrogen bond, and electrostatic interactions. Although sometimes regarded as nonspecific, electrostatic interactions are important in this optimization because the specific position of the phosphates may allow for specific charge-charge interactions with bound ligands. In this work, we have investigated the contribution of electrostatic interactions to the binding affinity of aminoglycoside antibiotics for TAR RNA. Because the charges in aminoglycoside antibiotics are provided by protonated amino groups, it is difficult to separate the contribution of hydrogen bonds and electrostatics to their binding specificity. Hence, we have investigated the dependence of the binding affinity on salt concentration, which should affect only the electrostatic contributions. Our results show that four aminoglycoside antibiotics (paromomycin, kanamycin-B, gentamycin, and tobramycin) bind TAR RNA with different affinities. Furthermore, the dependence of the binding affinity on salt concentration is different for kanamycin-B and paromomycin, with kanamycin-B showing a stronger dependence. Because all these antibiotics contain five positive charges, the results suggest that each antibiotic orients its charges in different ways when bound to TAR RNA. Our overall results support the idea that charge-charge interactions can contribute significantly to the specific binding of antibiotics to TAR RNA. Hence, the exact position of the charges should be considered in the design of any inhibitor of the interactions of TAR RNA.

Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca(2+) levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca(2+) sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca(2+) and GCAPs.