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DNA ligases are essential guardians of genome integrity by virtue of their ability to recognize and seal 3′-OH/5′-phosphate nicks in duplex DNA. The substrate binding and three chemical steps of the ligation pathway are coupled to global and local changes in ligase structure, involving both massive protein domain movements and subtle remodeling of atomic contacts in the active site. Here we applied solution NMR spectroscopy to study the conformational dynamics of the Chlorella virus DNA ligase (ChVLig), a minimized eukaryal ATP-dependent ligase consisting of nucleotidyltransferase, OB, and latch domains. Our analysis of backbone 15 N spin relaxation and 15 N, 1 H residual dipolar couplings of the covalent ChVLig-AMP intermediate revealed conformational sampling on fast (picosecond to nanosecond) and slow timescales (microsecond to millisecond), indicative of interdomain and intradomain flexibility. We identified local and global changes in ChVLig-AMP structure and dynamics induced by phosphate. In particular, the chemical shift perturbations elicited by phosphate were clustered in the peptide motifs that comprise the active site. We hypothesize that phosphate anion mimics some of the conformational transitions that occur when ligase-adenylate interacts with the nick 5′-phosphate.

DNA ligases specifically recognize and seal double stranded nicked DNA by catalyzing the formation of a phospho-diester bond between the 3′OH and 5′ phosphate termini. In physiological conditions these ubiquitous enzymes are essential for DNA replication, repair and recombination, while in tumor cells they can play a critical role in apoptosis resistance.Chlorella virus ligase is a pluripotent ATP-dependent ligase composed by two domains, a N-terminal nucleotidyltransferase domain, hosting the catalytic site, and a C-terminal OB-domain, both of which participate to DNA binding. A number of crystallographic studies have elucidated important structural details of the nick-sealing process. DNA binding, in particular, appears to require a large reorientation of the two domains, as well as relevant structural rearrangements localized mainly in the N-terminal region (1). Indeed this protein appears to be a highly dynamic system whose internal motions are closely linked to both the DNA recognition and to the catalytic process. So far, however, the actual nature of these motions is still largely unknown, not only for Chlorella virus ligase, but also for the entire protein family.We therefore tried to close this gap by undertaking the analysis of the dynamic properties of Chlorella virus liagase by solution NMR spectroscopy.Reference:(1) Nair P.A., Nandakumar J., Smith P., Odell M., Lima C.D., Shuman S. (2008) Nat Struct Mol Biol. 14, 770-8.

The 147 nm photolysis of GeH 4 results in the formation of H 2 , Ge 2 H 6 , Ge 3 H 8 , Ge 4 H 10 and a solid hydridic germanium film or powder. Two primary processes are involved: (a) GeH 4 + hv → GeH 2 + 2H and (b) GeH 4 + hv → GeH 3 + H, with π a = 0.66 and π b = 0.34. The quantum yields depend rather strongly on the partial pressure of GeH 4 , but at 2 Torr are Φ(−GeH 4 ) = 9.0 ± 0.4 and Φ(Ge 2 H 6 ) = 2.6 ± 0.7. The quantum yields of Ge 3 H 8 and Ge 4 H 10 were not measured at 2 Torr, but the average values found at lower pressures were 0.10Φ(Ge 2 H 6 ) and 0.02Φ(Ge 2 H 6 ) respectively; Φ(H 2 ) was not measured. A mechanism is proposed, which incorporates the known reactions of hydrogen atoms, GeH 2 radicals and GeH 3 radicals with GeH 4 and each other, and is shown to be in reasonable accord with the experimental facts.

Accurate quantification of the 15N–{1H} steady-state NOE is central to current methods for the elucidation of protein backbone dynamics on the fast, sub-nanosecond time scale. This experiment is highly susceptible to systematic errors arising from multiple sources. The nature of these errors and their effects on the determined NOE ratio is evaluated by a detailed analysis of the spin dynamics during the pair of experiments used to measure this ratio and possible improvements suggested. The experiment that includes 1H irradiation, is analyzed in the framework of Average Liouvillian Theory and a modified saturation scheme that generates a stable steady-state and eliminates the need to completely saturate 1H nuclei is presented. The largest source of error, however, in 1H-dilute systems at ultra-high fields is found to be an overestimation of the steady-state NOE value as a consequence of the incomplete equilibration of the magnetization in the so-called “reference experiment”. The use of very long relaxation delays is usually an effective, but time consuming, solution. Here, we introduce an alternative reference experiment, designed for larger, deuterated systems, that uses the fastest relaxing component of the longitudinal magnetization as a closer approximation to the equilibrium state for shorter relaxation delays. The utility of the modified approach is illustrated through simulations on realistic spin systems over a wide range of time scales and experimentally verified using a perdeuterated sample of human ubiquitin.

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated DeltaF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL.CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.

The family of multidomain proteins known as the synaptic associated proteins (SAPs) act as molecular scaffolds, playing an important role in the signaling and maintenance of several receptors and channels. The SAPs consist of 5 individual protein domains: 3 PDZ (PSD95, Disc Large, Zo1) domains, an SH3 domain, and an inactive guanyl kinase (GK) domain. The 3 PDZ domains bind the C-termini of specific receptors and channels, leading to the transient association with cytoskeletal and signaling proteins. Molecules targeting specific domains of the SAPs may provide a novel route for the regulation of channel and receptor function. Here we describe a structural-based approach for the development of such inhibitors for the PDZ domains of SAP90. The high sequence homology of the 3 domains has necessitated targeting regions outside the canonical binding pocket. The structural features of the PDZ domains with the C-termini of different receptors (GluR6), channels (Kv1.4), and cytoskeletal proteins (CRIPT) provide insight into targeting these regions.

The structural complexity of small peptides is such that the use of even a limited cassette of natural and unnatural amino acids in the development of structure-activity relationships can result in potential target arrays of discrete compounds. Such diversity is a logistic challenge, even in an era where parallel synthesis and high throughput assay methods are widely practiced. We have therefore concentrated on the development of small “focused” arrays, and the challenge becomes one of identifying the site(s) of focus. The somatostatin (ss) agonist BIM-23023 [1], and the recently described somatostatin antagonist BIM-23454 [2], have modest selectivity for hSSTR2 and we were interested in exploring the relationship between structure and function with respect to affinity for, and efficacy at alternative somatostatin receptor subtypes.

Over the last few years the importance of the intracellular C-terminus in the signaling of G-protein coupled receptors (GPCR) has become increasingly evident. In an effort to provide a structural framework for biological function, we have determined the conformation of the C-terminus of the bradykinin (BK) B2 receptor. Using a uniformly 15N- and 13C-enriched sample of the BKB2 receptor [309–366], NMR results clearly define three α-helices lying on the zwitterionic surface of the dodecylphosphocholine. The proximal helix consisting of residues 311–326 was previously predicted based on homology modeling with rhodopsin. This corresponds to what is often called helix-8 of the GPCRs. The two distal helices, residues 333–345 and 348–363, are clearly borne out by the NMR data. The functional importance of these secondary structural elements was probed by determination of the signaling properties (inositol phosphate formation) of mutant BKB2 receptors lacking the domains (deletion mutants) or containing the corresponding region from the related GPCR, angiotensin II AT1a (chimera receptors). We demonstrate that the regions between the helices (residues 327–333 and 346–347) can be exchanged without loss of signaling. In contrast, modification of the three helices, particularly the hydroxyl-containing residues, has drastic effects on the signaling profile of the BKB2 receptor. By coupling of the structural features with the functional data, the molecular mechanisms of signaling by the BKB2 receptor are beginning to be established.© 2005 Wiley Periodicals, Inc. Biopolymers (Pept Sci), 2005

Residue 19 of the parathyroid hormone (PTH) has been shown to play an important role in both binding to and activation of the PTH receptor; specifically, Arg(19)-containing analogues have improved biological function over similar Glu(19) peptides [Shimizu et al. (2002) Biochemistry 41, 13224-13233]. Additionally the juxtamembrane portion of the receptor is involved in the different biological responses. Here, we determine the conformational preferences of PTH analogues to provide a structural basis for their biological actions. On the basis of circular dichroism results, the Arg(19) --Glu(19) mutations within the context of both PTH(1-20) and PTH(1-34) analogues lead to increases in helix content, ranging from a 8-15% increase. High-resolution structures as determined by (1)H NMR and NOE-restrained molecular dynamics simulations clearly illustrate the difference between Arg(19) and Glu(19)-PTH(1-20), particularly with the extent and stability of the C-terminal helix. The Arg(19)-containing analogue has a well defined, stable alpha-helix from Ser(4)-Arg(19), while the Glu(19) analogue is less ordered at the C-terminus. On the basis of these observations, we propose that position 19 of PTH(1-20) must be alpha-helical for optimal interaction with the juxtamembrane portion of the receptor. This mode of binding extends the current view of PTH binding (indeed ligand binding for all class B GPCRs), which invokes a bihelical ligand with the C-terminus of the ligand interacting with the N-terminus of the receptor (responsible for binding) and the N-terminus of the ligand interacting with the seven-helical bundle (leading to receptor activation).

The intracellular (IC) face of the G-protein coupled receptors (GPCR), bradykinin (BK) B2 and angiotensin (AT) 1a, is similar in sequence homology and in size. Both receptors are known to link to Galphai and Galphaq but differ markedly in a number of physiologic actions, particularly with respect to their hemodynamic action. We made single as well as multiple, global replacements within the IC of BKB2R with the corresponding regions of the AT1aR. When stably transfected into Rat-1 cells, these hybrid receptors all bound BK with high affinity. Single replacement of the intracellular loop 2 (IC2) or the distal 34 residues of the C-terminus (dCt) with the corresponding regions of AT1aR resulted in chimera, which turned over phosphotidylinositol (PI) and released arachidonic acid (ARA) as WT BKB2R. In contrast, incorporation of the AT1aR IC3 in a single replacement abolished signal transduction. However, the simultaneous exchange of IC2 and IC3 of BKB2R with AT1aR resulted in a receptor responding to BK with PI turnover and ARA release approximately 4-fold greater than WT BKB2R. Likewise, the simultaneous replacement of IC2 and dCt resulted in a 2.8- and 1.6-fold increase in PI turnover and ARA release, respectively. In contrast, the dual replacement of IC3 and dCt could not overcome the deleterious effects of the IC3 replacement, resulting in very low PI activation and ARA release. Replacement of all three IC domains (IC2, IC3, and dCt) resulted in PI closer to that of AT1aR than BKB2R. The uptake of the receptor chimeras was similar to that of WT BKB2R with the exception of the IC3/dCt dual mutant, which exhibited very poor internalization (18% at 60'). When transfected into Rat-1 cells, the AT1aR markedly increased the expression of connective tissue growth factor (CTGF) mRNA, while BK slightly decreased it. The dual IC2/dCt and triple IC2/IC3/dCt hybrids both upregulated CTGF mRNA in response to BK. These results show that the IC face of the BKB2R can be exchanged with that of AT1aR, producing hybrid receptors, which take on the functional characteristics of AT1aR. The characterization of the chimera with stepwise replacement of the IC domains should allow for assignment of specific roles to the individual loops and C-terminus in the signaling and internalization of the BKB2R and facilitate the generation of a receptor with BKB2R binding and AT1aR function.

We report a new, higher resolution NMR structure of alpha-bungarotoxin that defines the structure-determining disulfide core and beta-sheet regions. We further report the NMR structure of the stoichiometric complex formed between alpha-bungarotoxin and a recombinantly expressed 19-mer peptide ((178)IPGKRTESFYECCKEPYPD(196)) derived from the alpha7 subunit of the chick neuronal nicotinic acetylcholine receptor. A comparison of these two structures reveals binding-induced stabilization of the flexible tip of finger II in alpha-bungarotoxin. The conformational rearrangements in the toxin create an extensive binding surface involving both sides of the alpha7 19-mer hairpin-like structure. At the contact zone, Ala(7), Ser(9), and Ile(11) in finger I and Arg(36), Lys(38), Val(39), and Val(40) in finger II of alpha-bungarotoxin interface with Phe(186), Tyr(187), Glu(188), and Tyr(194) in the alpha7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the alpha7 receptor, places alpha-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site. The disulfide-rich core of alpha-bungarotoxin is suggested to be tilted in the direction of the membrane surface with finger II extending into the proposed ligand-binding cavity.

The structural features of the PDZ1 domain of the synapse-associated protein SAP90 have been characterized by NMR. A comparison with the structures of the PDZ2 and PDZ3 domains of SAP90 illustrates significant differences, which may account for the unique binding properties of these homologous domains. Within the postsynaptic density, SAP90 functions as a molecular scaffold with a number of the protein-protein interactions mediated through the PDZ1 domain. Here, using fluorescence anisotropy and NMR chemical shift analysis, we have characterized the association of PDZ1 to the C-terminal peptides of the GluR6 subunit of the kainate receptor, voltage-gated K(+) channel Kv1.4, and microtubule-associate protein CRIPT, all of which are known to associate with SAP90. The latter two, which possess the consensus sequence for binding to PDZ domains (T/S-X-V-oh), have low micromolar binding affinities (1.5-15 microm). The C terminus of GluR6, RLPGKETMA-oh, lacking the consensus sequence, binds to PDZ1 of SAP90 with an affinity of 160 microm. The NMR data illustrate that although all three peptides occupy the binding groove capped by the GLGF loop of PDZ1, specific differences are present, consistent with the variation in binding affinities.

We have previously described the structural features of parathyroid hormone (PTH) as determined by high-resolution NMR under a number of different conditions, including the membrane mimetic environment of dodecylphosphocholine (DPC) micelles [1]. The structure of PTH consisted of a C-terminal helix extending from Glu19-Asn33, which is important for binding, and a N-terminal helix from residues Ser3-Asn10, which is responsible for activation of the PTH receptor. Based on a series of titrations with a relaxation-inducing lipid, the N-terminal helix was observed to be closely associated with the membrane surface. To further examine the importance of the interaction of the N-terminus with the membrane surface, we have undertaken the structural characterization of PTH containing cyclohexylalanine (Cha) at positions 7 and 11, replacing the naturally occurring leucines at these positions. Here, we present the results from our NMR-based structural investigation of this PTH analog, [Cha7,11]hPTH(l-34)NH2.

The structural features of the first extracellular loop (ECL1) of the parathyroid hormone receptor (PTH1R) in the presence of dodecylphosphocholine micelles have been determined using high-resolution NMR techniques. The structure of the receptor fragment, PTH1R(241-285), includes three alpha-helices for residues 241-244, 256-264, and 275-284. The first and third correspond to the end and the beginning of transmembrane helices 2 and 3, respectively. Centrally located in the second helix is L(261), found to cross-link to Lys(27) of parathyroid hormone, PTH(1-34) [Greenberg, Z., Bisello, A., Mierke, D. F., Rosenblatt, M., and Chorev, M. (2000) Biochemistry 39, 8142-8152]. On the basis of nitroxide radical-induced relaxation studies, the central helix is found to associate with the surface of the membrane mimetic. These data, in conjunction with previous results indicating a preference of PTH for the lipid surface, suggest a membrane-associated pathway for the initial recognition and binding of PTH to its G-protein-coupled receptor. Using the structural features of ECL1 as determined here, along with the structure of the PTH(1-34), the intermolecular interactions consistent with the contact point between L(261)(receptor)-Lys(27)(ligand) are identified.

The recently identified natural peptide ligand, tuberoinfundibular peptide of 39 residues (TIP39) for the parathyroid hormone-2 (PTH2) receptor has been structurally characterized by high resolution NMR, circular dichroism, and computer simulations. The structural features of TIP39, determined in the presence of a zwitterionic lipid to mimic the membrane environment of the G-protein-coupled PTH2 receptor, consist of two alpha-helices, Ala(5)-Arg(21) and Leu(26)-Val(35). Although TIP39 shares limited sequence homology with parathyroid hormone (PTH), a comparison of the structural features of TIP39 and PTH illustrates a similar topological display of residues of the N-terminal helix important for PTH2 receptor activation. The C-terminal helix of TIP39 differs from that of PTH with respect to size and amphipathicity, suggesting an altered mode of binding for TIP39, consistent with the receptor chimera and ligand truncation studies presented in the accompanying paper (Hoare, S. R. J., Clark, J. A., and Usdin, T. B. (2000) J. Biol. Chem. 275, 27274-27283). The structural characterization of TIP39 also provides some insight into the lack of affinity of this novel ligand for the PTH1 receptor.

The recently identified natural peptide ligand, tuberoinfundibular peptide of 39 residues (TIP39) for the parathyroid hormone-2 (PTH2) receptor has been structurally characterized by high resolution NMR, circular dichroism, and computer simulations. The structural features of TIP39, determined in the presence of a zwitterionic lipid to mimic the membrane environment of the G-protein-coupled PTH2 receptor, consist of two α-helices, Ala5–Arg21 and Leu26–Val35. Although TIP39 shares limited sequence homology with parathyroid hormone (PTH), a comparison of the structural features of TIP39 and PTH illustrates a similar topological display of residues of the N-terminal helix important for PTH2 receptor activation. The C-terminal helix of TIP39 differs from that of PTH with respect to size and amphipathicity, suggesting an altered mode of binding for TIP39, consistent with the receptor chimera and ligand truncation studies presented in the accompanying paper (Hoare, S. R. J., Clark, J. A., and Usdin, T. B. (2000) J. Biol. Chem. 275, 27274–27283). The structural characterization of TIP39 also provides some insight into the lack of affinity of this novel ligand for the PTH1 receptor.

The mechanisms by which MAP kinases recognize and phosphorylate substrates are not completely understood. Efforts to understand the mechanisms have been compromised by the lack of MAPK-substrate structures. While MAPK-substrate docking is well established as a viable mechanism for bringing MAPKs and substrates into close proximity the molecular details of how such docking promotes phosphorylation is an unresolved issue. In the present study computer modeling approaches, with restraints derived from experimentally known interactions, were used to predict how the N-terminus of Ets-1 associates with ERK2. Interestingly, the N-terminus does not contain a consensus-docking site ((R/K)(2-3)-X(2-6)-Φ(A)-X-Φ(B), where Φ is aliphatic hydrophobic) for ERK2. The modeling predicts that the N-terminus of Ets-1 makes important contributions to the stabilization of the complex, but remains largely disordered. The computer-generated model was used to guide mutagenesis experiments, which support the notion that Leu-11 and possibly Ile-13 and Ile-14 of Ets-1 1-138 (Ets) make contributions through binding to the hydrophobic groove of the ERK2 D-recruiting site (DRS). Based on the modeling, a consensus-docking site was introduced through the introduction of an arginine at residue 7, to give the consensus (7)RK-X(2)-Φ(A)-X-Φ(B) (13). This results in a 2-fold increase in k(cat)/K(m) for the phosphorylation of Ets by ERK2. Similarly, the substitution of the N-terminus for two different consensus docking sites derived from Elk-1 and MKK1 also improves k(cat)/K(m) by two-fold compared to Ets. Disruption of the N-terminal docking through deletion of residues 1-23 of Ets results in a 14-fold decrease in k(cat)/K(m), with little apparent change in k(cat). A peptide that binds to the DRS of ERK2 affects K(m), but not k(cat). Our kinetic analysis suggests that the unstructured N-terminus provides 10-fold uniform stabilization of the ground state ERK2•Ets•MgATP complex and intermediates of the enzymatic reaction.