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Maltodextrin glucosidase (MalZ) is a key enzyme in the maltose utilization pathway in Escherichia coli that liberates glucose from the reducing end of the short malto-oligosaccharides. Unlike other enzymes in the GH13_21 subfamily, the hydrolytic activity of MalZ is limited to maltodextrin rather than long starch substrates, forming various transglycosylation products in α-1,3, α-1,4 or α-1,6 linkages. The mechanism for the substrate binding and hydrolysis of this enzyme is not well understood yet. Here, we present the dimeric crystal structure of MalZ, with the N-domain generating a unique substrate binding groove. The N-domain bears CBM34 architecture and forms a part of the active site in the catalytic domain of the adjacent molecule. The groove found between the N-domain and catalytic domain from the adjacent molecule, shapes active sites suitable for short malto-oligosaccharides, but hinders long stretches of oligosaccharides. The conserved residue of E44 protrudes at subsite +2, elucidating the hydrolysis pattern of the substrate by the glucose unit from the reducing end. The structural analysis provides a molecular basis for the substrate specificity and the enzymatic property, and has potential industrial application for protein engineering.

The marine dinoflagellate Alexandrium is associated with harmful algal blooms (HABs) worldwide, causing paralytic shellfish poisoning (PSP) in humans. We found that the marine bacterium Pseudoruegeria sp. M32A2M exhibits algicidal activity against Alexandrium catenella (Group I), inhibiting its motility and consequently inducing cell disruption after 24 h of co-culture. To understand the communication between the two organisms, we investigated the time-course cellular responses through genome-wide transcriptome analysis. Functional analysis of differentially expressed genes revealed that the core reactions of the photosystem in A. catenella were inhibited within 2 h, eventually downregulating the entire pathways of oxidative phosphorylation and carbon fixation, as well as associated metabolic pathways. Conversely, Pseudoruegeria upregulated its glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation pathways. Also, the transporters for nutrients such as C3/C4 carbohydrates and peptides were highly upregulated, leading to the speculation that nutrients released by disrupted A. catenella cells affect the central metabolism of Pseudoruegeria. In addition, we analyzed the secondary metabolite-synthesizing clusters of Pseudoruegeria that were upregulated by co-culture, suggesting their potential roles in algicidal activity. Our time-course transcriptome analysis elucidates how A. catenella is affected by algicidal bacteria and how these bacteria obtain functional benefits through metabolic pathways.

Intracellular antibodies have become powerful tools for imaging, modulating and neutralizing endogenous target proteins. Here, we describe an optogenetically activated intracellular antibody (optobody) consisting of split antibody fragments and blue-light inducible heterodimerization domains. We expanded this optobody platform by generating several optobodies from previously developed intracellular antibodies, and demonstrated that photoactivation of gelsolin and β2-adrenergic receptor (β2AR) optobodies suppressed endogenous gelsolin activity and β2AR signaling, respectively.

Contrary to the general concept, the archaeal Smc-based complex appears to lack the kite subunit ScpB that is essential in the bacterial Smc–ScpAB complex that mediates chromosome organization., SMC complexes play a central role in chromosome organization in all domains of life. The bacterial Smc–ScpAB complex is a three-subunit complex composed of Smc, ScpA and ScpB. ScpA bridges the two ATPase domains of the Smc homodimer, while ScpB, which belongs to the kite family of proteins, interacts with ScpA. The three subunits are known to be equally important for the function of Smc–ScpAB in bacteria. From crystallographic and biochemical studies, evidence is provided that six archaeal ScpA proteins are unable to interact with the only putative ScpB found in these species. Structure-based sequence alignment reveals that these archaeal ScpAs lack the ScpB-binding segment that is commonly present in the middle of bacterial ScpA sequences, which is thus responsible for their inability to interact with ScpB. ScpA proteins lacking the ScpB-binding segment are found to prevail in archaea. Moreover, two archaeal ScpA proteins with a longer middle region also failed to bind their putative ScpB partner. Furthermore, all or most species belonging to five out of 14 euryarchaeotal orders contain Smc and ScpA but not a detectable ScpB homologue. These data support the notion that archaeal Smc-based complexes generally function as a two-subunit complex composed of only Smc and ScpA.

Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site–coordinating core β-sheet–containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.

Protein tyrosine phosphatases (PTPs) originating from eukaryotes or bacteria have been under intensive structural and biochemical investigation, whereas archaeal PTP proteins have not been investigated extensively; therefore, they are poorly understood. Here, we present the crystal structures of Tk-PTP derived from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1, in both the active and inactive forms. Tk-PTP adopts a common dual-specificity phosphatase (DUSP) fold, but it undergoes an atypical temperature-dependent conformational change in its P-loop and α4−α5 loop regions, switching between the inactive and active forms. Through comprehensive analyses of Tk-PTP, including additional structural determination of the G95A mutant form, enzymatic activity assays, and structural comparison with the other archaeal PTP, it was revealed that the presence of the GG motif in the P-loop is necessary but not sufficient for the structural flexibility of Tk-PTP. It was also proven that Tk-PTP contains dual general acid/base residues unlike most of the other DUSP proteins, and that both the residues are critical in its phosphatase activity. This work provides the basis for expanding our understanding of the previously uncharacterized PTP proteins from archaea, the third domain of living organisms.

SummaryThe CRISPR-Cas system is the RNA-guided immune defense mechanism in bacteria and archaea. Csm1 belongs to the Cas10 family, which is the common signature protein of the type III system. Csm1 is the largest subunit of the Csm interference complex in the type III-A subtype, which targets foreign DNA or RNA. Here, we report crystallographic and biochemical analyses of Thermococcus onnurineus Csm1, revealing a five-domain organization and single-stranded DNA (ssDNA)-specific nuclease activity associated with the N-terminal HD domain. This domain folds into permuted secondary structures in comparison with the HD domain of Cas3 and contains all the catalytically important residues. It exhibited both endo- and exonuclease activities in an Ni2+ or Mn2+-dependent manner. The narrow width of the active-site cleft appears to restrict the substrate specificity to ssDNA and thus to prevent Csm1 from cleaving double-stranded chromosomal DNA. These data suggest that Csm1 may function in DNA interference by the Csm effector complex.

Human papillomaviruses (HPVs) are causative agents of various diseases associated with cellular hyperproliferation, including cervical cancer, one of the most prevalent tumors in women. E7 is one of the two HPV-encoded oncoproteins and directs recruitment and subsequent degradation of tumor-suppressive proteins such as retinoblastoma protein (pRb) via its LxCxE motif. E7 also triggers tumorigenesis in a pRb-independent pathway through its C-terminal domain, which has yet been largely undetermined, with a lack of structural information in a complex form with a host protein. Herein, we present the crystal structure of the E7 C-terminal domain of HPV18 belonging to the high-risk HPV genotypes bound to the catalytic domain of human nonreceptor-type protein tyrosine phosphatase 14 (PTPN14). They interact directly and potently with each other, with a dissociation constant of 18.2 nM. Ensuing structural analysis revealed the molecular basis of the PTPN14-binding specificity of E7 over other protein tyrosine phosphatases and also led to the identification of PTPN21 as a direct interacting partner of E7. Disruption of HPV18 E7 binding to PTPN14 by structure-based mutagenesis impaired E7’s ability to promote keratinocyte proliferation and migration. Likewise, E7 binding-defective PTPN14 was resistant for degradation via proteasome, and it was much more effective than wild-type PTPN14 in attenuating the activity of downstream effectors of Hippo signaling and negatively regulating cell proliferation, migration, and invasion when examined in HPV18-positive HeLa cells. These results therefore demonstrated the significance and therapeutic potential of the intermolecular interaction between HPV E7 and host PTPN14 in HPV-mediated cell transformation and tumorigenesis., Human papillomaviruses cause various diseases associated with cellular hyperproliferation, including cervical cancer. Structural, biochemical, and cellular analyses reveal the molecular basis and significance of the intermolecular interaction between the E7 protein of human papillomavirus 18 and the human tumor suppressor protein PTPN14.

The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from Thermococcus onnurineus is reconstituted with a minimalistic combination of Csm1121334151, and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ssDNA containing a sequence complementary to the bound crRNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ssDNA without a target RNA suggests a novel ssDNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.

A hypothetical protein TON_0340 of a Thermococcus species is a protein conserved in a variety of organisms including human. Herein, we present four different crystal structures of TON_0340, leading to the identification of an active-site cavity harboring a metal-binding site composed of six invariant aspartate and glutamate residues that coordinate one to three metal ions. Biochemical and mutational analyses involving many phosphorous compounds show that TON_0340 is a Mn2+-dependent phosphatase. Mg2+ binds to TON_0340 less tightly and activates the phosphatase activity less efficiently than Mn2+. Whereas Ca2+ and Zn2+ are able to bind to the protein, they are unable to activate its enzymatic activity. Since the active-site cavity is small and largely composed of nearly invariant stretches of 11 or 13 amino acids, the physiological substrates of TON_0340 and its homologues are likely to be a small and the same molecule. The Mn2+-bound TON_0340 structure provides a canonical model for the ubiquitously present TON_0340 homologues and lays a strong foundation for the elucidation of their substrate and biological function.

Protrudin is a FYVE (Fab 1, YOTB, Vac 1, and EEA1) domain-containing protein involved in transport of neuronal cargoes and implicated in the onset of hereditary spastic paraplegia. Our image-based screening of the lipid binding domain library revealed novel plasma membrane localization of the FYVE domain of protrudin unlike canonical FYVE domains that are localized to early endosomes. The membrane binding study by surface plasmon resonance analysis showed that this FYVE domain preferentially binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)), phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P(2)), and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)) unlike canonical FYVE domains that specifically bind phosphatidylinositol 3-phosphate (PtdIns(3)P). Furthermore, we found that these phosphoinositides (PtdInsP) differentially regulate shuttling of protrudin between endosomes and plasma membrane via its FYVE domain. Protrudin mutants with reduced PtdInsP-binding affinity failed to promote neurite outgrowth in primary cultured hippocampal neurons. These results suggest that novel PtdInsP selectivity of the protrudin-FYVE domain is critical for its cellular localization and its role in neurite outgrowth.

In many prokaryotic organisms, chromosomal loci known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (CAS) genes comprise an acquired immune defense system against invading phages and plasmids. Although many different Cas protein families have been identified, the exact biochemical functions of most of their constituents remain to be determined. In this study, we report the crystal structure of PF1127, a Cas protein of Pyrococcus furiosus DSM 3638 that is composed of 480 amino acids and belongs to the Csx1 family. The C-terminal domain of PF1127 has a unique β-hairpin structure that protrudes out of an α-helix and contains several positively charged residues. We demonstrate that PF1127 binds double-stranded DNA and RNA and that this activity requires an intact β-hairpin and involve the homodimerization of the protein. In contrast, another Csx1 protein from Sulfolobus solfataricus P2 that is composed of 377 amino acids does not have the β-hairpin structure and exhibits no DNA-binding properties under the same experimental conditions. Notably, the C-terminal domain of these two Csx1 proteins is greatly diversified, in contrast to the conserved N-terminal domain, which appears to play a common role in the homodimerization of the protein. Thus, although P. furiosus Csx1 is identified as a nucleic acid-binding protein, other Csx1 proteins are predicted to exhibit different individual biochemical activities. Proteins 2013. © 2012 Wiley Periodicals, Inc.

Many prokaryotic organisms acquire immunity against foreign genetic material by incorporating a short segment of foreign DNA called spacer into chromosomal loci, termed clustered regularly interspaced short palindromic repeats (CRISPRs). The encoded RNAs are processed into small fragments that guide the silencing of the invading genetic elements. The CRISPR-associated (Cas) proteins are the main executioners of these processes. Herein, we report the crystal structure of Stu0660 of Streptococcus thermophilus, a Cas protein involved in the acquisition of new spacers. By homotetramerization, Stu0660 forms a central channel which is decorated with basic amino acids and binds linear double-stranded DNA (dsDNA), but not circular dsDNA. Despite undetectably low sequence similarity, two N-terminal domains of Stu0660 are similar to the entire structure of an Enterococcus faecalis Csn2 protein, which also forms a homotetramer and binds dsDNA. Thus, this work identifies a previously unknown group of Stu0660-like Csn2 proteins (∼350 residues), which are larger than the known canonical Csn2 proteins (∼220 residues) by containing an extra C-terminal domain. The commonly present central channel in the two subgroups appears as a design to selectively interact with linear dsDNA.

Staphylothermus marinus maltogenic amylase (SMMA) is a novel extreme thermophile maltogenic amylase with an optimal temperature of 100 °C, which hydrolyzes α-(1-4)-glycosyl linkages in cyclodextrins and in linear malto-oligosaccharides. This enzyme has a long N-terminal extension that is conserved among archaic hyperthermophilic amylases but is not found in other hydrolyzing enzymes from the glycoside hydrolase 13 family. The SMMA crystal structure revealed that the N-terminal extension forms an N' domain that is similar to carbohydrate-binding module 48, with the strand-loop-strand region forming a part of the substrate binding pocket with several aromatic residues, including Phe-95, Phe-96, and Tyr-99. A structural comparison with conventional cyclodextrin-hydrolyzing enzymes revealed a striking resemblance between the SMMA N' domain position and the dimeric N domain position in bacterial enzymes. This result suggests that extremophilic archaea that live at high temperatures may have adopted a novel domain arrangement that combines all of the substrate binding components within a monomeric subunit. The SMMA structure provides a molecular basis for the functional properties that are unique to hyperthermophile maltogenic amylases from archaea and that distinguish SMMA from moderate thermophilic or mesophilic bacterial enzymes.

GDP-bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub-cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP-to-GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA-1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure-based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.

The structural maintenance of chromosomes (SMC) family proteins are commonly found in the multiprotein complexes involved in chromosome organization, including chromosome condensation and sister chromatid cohesion. These proteins are characterized by forming a V-shaped homo- or heterodimeric structure with two long coiled-coil arms having two ATPase head domains at the distal ends. The hinge domain, located in the middle of the coiled coil, forms the dimer interface. In addition to being the dimerization module, SMC hinges appear to play other roles, including the gateway function for DNA entry into the cohesin complex. Herein, we report the homodimeric structure of the hinge domain of Escherichia coli MukB, which forms a prokaryotic condensin complex with two non-SMC subunits, MukE and MukF. In contrast with SMC hinge of Thermotoga maritima which has a sizable central hole at the dimer interface, MukB hinge forms a constricted dimer interface lacking a hole. Under our assay conditions, MukB hinge does not interact with DNA in accordance with the absence of a notable positively charged surface patch. The function of MukB hinge appears to be limited to dimerization of two copies of MukB molecules. Proteins 2010. © 2009 Wiley-Liss, Inc.

Condensin complexes are the key mediators of chromosome condensation. The MukB–MukE–MukF complex is a bacterial condensin, in which the MukB subunit forms a V-shaped dimeric structure with two ATPase head domains. MukE and MukF together form a tight complex, which binds to the MukB head via the C-terminal winged-helix domain (C-WHD) of MukF. One of the two bound C-WHDs of MukF is forced to detach from two ATP-bound, engaged MukB heads, and this detachment reaction depends on the MukF flexible linker preceding the C-WHD. Whereas MukB is known to focally localize at particular positions in cells by an unknown mechanism, mukE- or mukF-null mutation causes MukB to become dispersed in cells. Here, we report that mutations in MukF causing a defect in the detachment reaction interfere with the focal localization of MukB, and that the dispersed distribution of MukB in cells correlates directly with defects in cell growth and division. The data strongly suggest that the MukB–MukE–MukF condensin forms huge clusters through the ATP-dependent detachment reaction, and this cluster formation is critical for chromosome condensation by this machinery. We also show that the MukF flexible linker is involved in the dimerization and ATPase activity of the MukB head. Structured digital abstract • MINT-7216106: mukBhd (uniprotkb:P22523), mukF (uniprotkb:P60293) and mukE (uniprotkb:P22524) physically interact (MI:0915) by blue native page (MI:0276)

Chromosomes are condensed in all forms of life. SMC-based condensins are the key mediators in this process, but their molecular mechanisms remain elusive. Two different condensin complexes have been identified in prokaryotic organisms: MukB-MukE-MukF and SMC-ScpA-ScpB. This review focuses on comparison between the two machineries based on structural, biochemical and other related information in the light of their structure and function.

To elucidate the relationship between the substrate size and geometric shape of the catalytic site of Thermus maltogenic amylase, Gly50, Asp109, and Val431, located at the interface of the dimer, were replaced with bulky amino acids. The k(cat)/K(m) value of the mutant for amylose increased significantly, whereas that for amylopectin decreased as compared to that of the wild-type enzyme. Thus, the substituted bulky amino acid residues modified the shape of the catalytic site, such that the ability of the enzyme to distinguish between small and large molecules like amylose and amylopectin was enhanced.

Recognition of lysine-type peptidoglycan by peptidoglycan recognition protein (PGRP)-SA provokes the activation of the Toll and prophenoloxidase pathways. Here we reveal that a soluble fragment of lysine-type peptidoglycan, a long glycan chain with short stem peptides, is a potent activator of the Drosophila Toll pathway and the prophenoloxidase activation cascade in the beetle Tenebrio molitor . Using this peptidoglycan fragment, we present biochemical evidence that clustering of PGRP-SA molecules on the peptidoglycan is required for the activation of the prophenoloxidase cascade. We subsequently highlight that the lysozyme-mediated partial digestion of highly cross-linked lysine-type peptidoglycan dramatically increases the binding of PGRP-SA, presumably by inducing clustering of PGRP-SA, which then recruits the Gram-negative bacteria-binding protein 1 homologue and a modular serine protease containing low-density lipoprotein and complement control protein domains. The crucial role of lysozyme in the prophenoloxidase activation cascade is further confirmed in vivo by using a lysozyme inhibitor. Taken together, we propose a model whereby lysozyme presents a processed form of lysine-type peptidoglycan for clustering of PGRP-SA that recruits Gram-negative bacteria-binding protein 1 and the modular serine protease, which leads to the activation of both the Toll and prophenoloxidase pathways.

Malonamidase E2 (MAE2) from Bradyrhizobium japonicum is an enzyme that hydrolyzes malonamate to malonate and has a Ser-cis-Ser-Lys catalytic triad at the active site. The crystal structures of wild type and mutant MAE2 exhibited that the guanido group of Arg-158 could be involved in the binding of malonamate in which the negative charge of the carboxyl group could destabilize a negatively charged transition-state oxyanion in the enzymatic reaction. In an attempt to elucidate the specific roles of Arg-158, site-directed mutants, R158Q, R158E, and R158K, were prepared (see Table 1). The crystal structure of R158Q determined at 2.2 Angstrom resolution showed that the guanido group of Arg-158 was important for the substrate binding with the marginal structural change upon the mutation. The k(cat) value of R158Q significantly decreased by over 1500-fold and the catalytic activity of R158E could not be detected. The k(cat) value of R158K was similar to that of the wild type with the K(m) value drastically increased by 100-fold, suggesting that Lys-158 of R158K can stabilize the negative charge of the carboxylate in the substrate to some extent and contribute to the stabilization of the transition-state oxyanion, but a single amine group of Lys-158 in R158K could not precisely anchor the carboxyl group of malonamate compared with the guanido group of Arg-158. Our kinetic and structural evidences demonstrate that Arg-158 in MAE2 should be critical to both binding the substrate and stabilizing the transition-state oxyanion for the catalytic reaction of MAE2.

Transport protein particle (TRAPP) comprises a family of two highly related multiprotein complexes, with seven common subunits, that serve to target different classes of transport vesicles to their appropriate compartments. Defining the architecture of the complexes will advance our understanding of the functional differences between these highly related molecular machines. Genetic analyses in yeast suggested a specific interaction between the TRAPP subunits Bet3p and Trs33p. A mammalian bet3–trs33 complex was crystallized, and the structure was solved to 2.2 A resolution. Intriguingly, the overall fold of the bet3 and trs33 monomers was similar, although the proteins had little overall sequence identity. In vitro experiments using yeast TRAPP subunits indicated that Bet3p binding to Trs33p facilitates the interaction between Bet3p and another TRAPP subunit, Bet5p. Mutational analysis suggests that yeast Trs33p facilitates other Bet3p protein–protein interactions. Furthermore, we show that Trs33p can increase the Golgi-localized pool of a mutated Bet3 protein normally found in the cytosol. We propose that one of the roles of Trs33p is to facilitate the incorporation of the Bet3p subunit into assembling TRAPP complexes.

A structural motif called the small exterior hydrophobic cluster (SEHC) has been proposed to explain the stabilizing effect mediated by solvent-exposed hydrophobic residues; however, little is known about its biological roles. Unusually, in Δ5-3-ketosteroid isomerase from Pseudomonas putida biotype B (KSI-PI) Trp92 is exposed to solvent on the protein surface, forming a SEHC with the side-chains of Leu125 and Val127. In order to identify the role of the SEHC in KSI-PI, mutants of those amino acids associated with the SEHC were prepared. The W92A, L125A/V127A, and W92A/L125A/V127A mutations largely decreased the conformational stability, while the L125F/V127F mutation slightly increased the stability, indicating that hydrophobic packing by the SEHC is important in maintaining stability. The crystal structure of W92A revealed that the decreased stability caused by the removal of the bulky side-chain of Trp92 could be attributed to the destabilization of the surface hydrophobic layer consisting of a solvent-exposed β-sheet. Consistent with the structural data, the binding affinities for three different steroids showed that the surface hydrophobic layer stabilized by SEHC is required for KSI-PI to efficiently recognize hydrophobic steroids. Unfolding kinetics based on analysis of the ΦU value also indicated that the SEHC in the native state was resistant to the unfolding process, despite its solvent-exposed site. Taken together, our results demonstrate that the SEHC plays a key role in the structural integrity that is needed for KSI-PI to stabilize the hydrophobic surface conformation and thereby contributes both to the overall conformational stability and to the binding of hydrophobic steroids in water solution.

RbsD is the only protein whose biochemical function is unknown among the six gene products of the rbs operon involved in the active transport of ribose. FucU, a paralogue of RbsD conserved from bacteria to human, is also the only protein whose function is unknown among the seven gene products of the l-fucose regulon. Here we report the crystal structures of Bacillus subtilis RbsD, which reveals a novel decameric toroidal assembly of the protein. Nuclear magnetic resonance and other studies on RbsD reveal that the intersubunit cleft of the protein binds specific forms of d-ribose, but it does not have an enzyme activity toward the sugar. Likewise, FucU binds l-fucose but lacks an enzyme activity toward this sugar. We conclude that RbsD and FucU are cytoplasmic sugar-binding proteins, a novel class of proteins whose functional role may lie in helping influx of the sugar substrates.

Amidase signature family enzymes, which are widespread in nature, contain a newly identified Ser-cisSer-Lys catalytic triad in which the peptide bond between Ser131 and the preceding residue Gly130 is in a cis configuration. In order to characterize the property of the novel triad, we have determined the structures of five mutant malonamidase E2 enzymes that contain a Cys-cisSer-Lys, Ser-cisAla-Lys, or Ser-cisSer-Ala triad or a substitution of Gly130 with alanine. Cysteine cannot replace the role of Ser155 due to a hyper-reactivity of the residue, which results in the modification of the cysteine to cysteinyl sulfinic acid, most likely inside the expression host cells. The lysine residue plays a structural as well as a catalytic role, since the substitution of the residue with alanine disrupts the active site structure completely. The two observations are in sharp contrast with the consequences of the corresponding substitutions in the classical Ser-His-Asp triad. Structural data on the mutant containing the Ser-cisAla-Lys triad convincingly suggest that Ser131 plays an analogous catalytic role as the histidine of the Ser-His-Asp triad. The unusual cis configuration of Ser131 appears essential for the precise contacts of this residue with the other triad residues, as indicated by the near invariance of the preceding glycine residue (Gly130), structural data on the G130A mutant, and by a modeling experiment. The data provide a deep understanding of the role of each residue of the new triad at the atomic level and demonstrate that the new triad is a catalytic device distinctively different from the classical triad or its variants.

SEDL is an evolutionarily highly conserved protein in eukaryotic organisms. Deletions or point mutations in the SEDL gene are responsible for the genetic disease spondyloepiphyseal dysplasia tarda (SEDT), an X-linked skeletal disorder. SEDL has been identified as a component of the transport protein particle (TRAPP), critically involved in endoplasmic reticulum-to-Golgi vesicle transport. Herein, we report the 2.4 A resolution structure of SEDL, which reveals an unexpected similarity to the structures of the N-terminal regulatory domain of two SNAREs, Ykt6p and Sec22b, despite no sequence homology to these proteins. The similarity and the presence of unusually many solvent-exposed apolar residues of SEDL suggest that it serves regulatory and/or adaptor functions through multiple protein-protein interactions. Of the four known missense mutations responsible for SEDT, three mutations (S73L, F83S, V130D) map to the protein interior, where the mutations would disrupt the structure, and the fourth (D47Y) on a surface at which the mutation may abrogate functional interactions with a partner protein.

A novel inhibitor against maltose-producing alpha-amylase was prepared via stepwise degradation of a high-molecular-weight acarbose (HMWA) using Thermus maltogenic amylase (ThMA). The structure of the purified inhibitor was determined to be alpha-D-glucopyranosyl-alpha-acarviosinyl-D-glucopyranose (GlcAcvGlc) by (13)C NMR and MALDI-TOF/MS. Progress curves of PNPG2 hydrolysis by various amylolytic enzymes, including MGase, ThMA, and CDase I-5, in the presence of acarbose or GlcAcvGlc indicated a slow-binding mode of inhibition. Analytical ultracentrifugation and X-ray crystallography analyses revealed that the presence of GlcAcvGlc increased the dimerization of ThMA. The formation of dimer complexed with GlcAcvGlc might induce a conformational change in ThMA, leading to a two-step inhibition process. The inhibition potency of GlcAcvGlc for MGase, ThMA, and CDase I-5 was 3 orders of magnitude higher than that of acarbose.

A large group of hydrolytic enzymes, which contain a conserved stretch of approximately 130 amino acids designated the amidase signature (AS) sequence, constitutes a super family that is distinct from any other known hydrolase family. AS family enzymes are widespread in nature, ranging from bacteria to humans, and exhibit a variety of biological functions. Here we report the first structure of an AS family enzyme provided by the crystal structure of malonamidase E2 from Bradyrhizobium japonicum. The structure, representing a new protein fold, reveals a previously unidentified Ser-cisSer-Lys catalytic machinery that is absolutely conserved throughout the family. This family of enzymes appears to be evolutionarily distinct but has diverged to acquire a wide spectrum of individual substrate specificities, while maintaining a core structure that supports the catalytic function of the unique triad. Based of the structures of the enzyme in two different inhibited states, an unusual action mechanism of the triad is proposed that accounts for the role of the cis conformation in the triad.

Over 20 enzymes denoted as cyclomaltodextrinase, maltogenic amylase, or neopullulanase that share 40-86% sequence identity with each other are found in public data bases. These enzymes are distinguished from typical alpha-amylases by containing a novel N-terminal domain and exhibiting preferential substrate specificities for cyclomaltodextrins (CDs) over starch. In this research field, a great deal of confusion exists regarding the features distinguishing the three groups of enzymes from one another. Although a different enzyme code has been assigned to each of the three different enzyme names, even a single differentiating enzymatic property has not been documented in the literature. On the other hand, an outstanding question related to this issue concerns the structural basis for the preference of these enzymes for CDs. To clarify the confusion and to address this question, we have determined the structures of two enzymes, one from alkalophilic Bacillus sp. I-5 and named cyclomaltodextrinase and the other from a Thermus species and named maltogenic amylase. The structure of the Bacillus enzyme reveals a dodecameric assembly composed of six copies of the dimer, which is the structural and functional unit of the Thermus enzyme and an enzyme named neopullulanase. The structure of the Thermus enzyme in complex with beta-CD led to the conclusion that Trp47, a well conserved N-terminal domain residue, contributes greatly to the preference for beta-CD. The common dimer formation through the novel N-terminal domain, which contributes to the preference for CDs by lining the active-site cavity, convincingly indicates that the three groups of enzymes are not different enough to preserve the different names and enzyme codes.

Both d - and l -isomers of N -(hydroxyaminocarbonyl)phenylalanine ( 1 ) were shown to have strong binding affinity towards carboxypeptidase A (CPA) with d - 1 being more potent than its enantiomer by 3-fold (Chung, S. J.; Kim, D. H. Bioorg. Med. Chem. 2001 , 9 , 185.). In order to understand the reversed stereochemical preference shown in the CPA inhibition, we have solved the crystal structures of CPA complexed with each enantiometer of 1 up to 1.75 A resolution. Inhibitor l - 1 whose stereochemistry belongs to the stereochemical series of substrate binds CPA like substrate does with its carbonyl oxygen coordinating to the active site zinc ion. Its hydroxyl is engaged in hydrogen bonding with the carboxylate of Glu-270. On the other hand, in binding of d - 1 to CPA, its terminal hydroxyl group is involved in interactions with the active site zinc ion and the carboxylate of Glu-270. In both CPA· 1 complexes, the phenyl ring in 1 is fitted in the substrate recognition pocket at the S 1 ′ subsite, and the carboxylate of the inhibitors forms bifurcated hydrogen bonds with the guanidinium moiety of Arg-145 and a hydrogen bond with the guanidinium of Arg-127. In the complex of CPA· d - 1 , the carboxylate of the inhibitor is engaged in hydrogen bonding with the phenolic hydroxyl of the down-positioned Tyr-248. While the l - 1 binding induces a concerted movement of the backbone amino acid residues at the active site, only the downward movement of Tyr-248 was noted when d - 1 binds to CPA.

The thermostable phytase from Bacillus amyloliquefaciens DS11 hydrolyzes phytate (myo-inositol hexakisphosphate, IP6) to less phosphorylated myo-inositol phosphates in the presence of Ca2+. In this report, we discuss the unique Ca2+-dependent catalytic properties of the phytase and its specific substrate requirement. Initial rate kinetic studies of the phytase indicate that the enzyme activity follows a rapid equilibrium ordered mechanism in which binding of Ca2+ to the active site is necessary for the essential activation of the enzyme. Ca2+ turned out to be also required for the substrate because the phytase is only able to hydrolyze the calcium-phytate complex. In fact, both an excess amount of free Ca2+ and an excess of free phytate, which is not complexed with each other, can act as competitive inhibitors. The Ca2+-dependent catalytic activity of the enzyme was further confirmed, and the critical amino acid residues for the binding of Ca2+ and substrate were identified by site-specific mutagenesis studies. Isothermal titration calorimetry (ITC) was used to understand if the decreased enzymatic activity was related to poor Ca2+ binding. The pH dependence of the Vmax and Vmax/Km consistently supported these observations by demonstrating that the enzyme activity is dependent on the ionization of amino acid residues that are important for the binding of Ca2+ and the substrate. The Ca2+-dependent activation of enzyme and substrate was found to be different from other histidine acid phytases that hydrolyze metal-free phytate.

Helicobacter pylori, an etiologic agent in a variety of gastroduodenal diseases, produces a large amount of urease, which is believed to neutralize gastric acid by producing ammonia for the survival of the bacteria. Up to 30% of the enzyme associates with the surface of intact cells upon lysis of neighboring bacteria. The role of the enzyme at the extracellular location has been a subject of controversy because the purified enzyme is irreversibly inactivated below pH 5. We have determined the crystal structure of H. pylori urease, which has a 1.1 MDa spherical assembly of 12 catalytic units with an outer diameter of approximately 160 A. Under physiologically relevant conditions, the activity of the enzyme remains unaffected down to pH 3. Activity assays under different conditions indicated that the cluster of the 12 active sites on the supramolecular assembly may be critical for the survival of the enzyme at low pH. The structure provides a novel example of a molecular assembly adapted for acid resistance that, together with the low Km value of the enzyme, is likely to enable the organism to inhabit the hostile niche.

Δ5-3-ketosteroid isomerase (KSI) from Pseudomonas putida Biotype B catalyzes the allylic isomerization of Δ5-3-ketosteroids to their conjugated Δ4-isomers via a dienolate intermediate. Two electrophilic catalysts, Tyr-14 and Asp-99, are involved in a hydrogen bond network that comprises Asp-99 Oδ2···O of Wat504···Tyr-14 Oη···Tyr-55 Oη···Tyr-30 Oη in the active site of P. putida KSI. Even though neither Tyr-30 nor Tyr-55 plays an essential role in catalysis by the KSI, the catalytic activity of Y14F could be increased ca. 26−51-fold by the additional Y30F and/or Y55F mutation in the hydrogen bond network. To identify the structural basis for the pseudoreversion in the KSI, crystal structures of Y14F and Y14F/Y30F/Y55F have been determined at 1.8 and 2.0 A resolution, respectively. Comparisons of the two structures near the catalytic center indicate that the hydrogen bond between Asp-99 Oδ2 and C3−O of the steroid, which is perturbed by the Y14F mutation, can be partially restored to that in the wild-type e...

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.

Phytases hydrolyze phytic acid to less phosphorylated myo-inositol derivatives and inorganic phosphate. A thermostable phytase is of great value in applications for improving phosphate and metal ion availability in animal feed, and thereby reducing phosphate pollution to the environment. Here, we report a new folding architecture of a six-bladed propeller for phosphatase activity revealed by the 2.1 A crystal structures of a novel, thermostable phytase determined in both the partially and fully Ca2+-loaded states. Binding of two calcium ions to high-affinity calcium binding sites results in a dramatic increase in thermostability (by as much as ∼30°C in melting temperature) by joining loop segments remote in the amino acid sequence. Binding of three additional calcium ions to low-affinity calcium binding sites at the top of the molecule turns on the catalytic activity of the enzyme by converting the highly negatively charged cleft into a favorable environment for the binding of phytate.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp(38) (pK(a) approximately 4.7) was identified as the general base abstracting the steroid C4beta proton (pK(a) approximately 12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals clearly that both the Tyr(14) OH and Asp(99) COOH provide direct hydrogen bonds to the oxyanion of equilenin. The result negates the catalytic dyad mechanism in which Asp(99) donates the hydrogen bond to Tyr(14), which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr(14) OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate.

Electrospray ionization mass spectrometry (ESI-MS) is employed to directly analyze the limited trypsinolysis products of wild-type tumor necrosis factor-alpha (wtTNF-alpha) and its mutant, M3S. To determine the charge numbers of peaks of relatively small peptides in the ESI mass spectrum of a digest, a series of sodium-adduct ion peaks of each peptide are generated by adding a small quantity of NaCl to the digest before taking the spectrum. From the monitoring of the composition of proteolytic mixture as the incubation time is lengthened, it has been learned that the proteolysis of wtTNF-alpha by trypsin occurs sequentially: Arg2, Arg6, Arg32, Arg31, and Arg44, and that M3S is strongly resistant to the proteolysis. Since the cleavage sequence of wtTNF-alpha and the mutation-induced resistance of M3S are consistent with the structural features of the proteins, we can suggest a mutant more resistant to proteolysis than M3S, which has an additional point mutation, Ala35Leu or Ala35Ile.

Crystal structure of RNA helicase domain from genotype 1b hepatitis C virus has been determined at 2.3 A resolution by the multiple isomorphous replacement method. The structure consists of three domains that form a Y-shaped molecule. One is a NTPase domain containing two highly conserved NTP binding motifs. Another is an RNA binding domain containing a conserved RNA binding motif. The third is a helical domain that contains no β-strand. The RNA binding domain of the molecule is distinctively separated from the other two domains forming an interdomain cleft into which single stranded RNA can be modeled. A channel is found between a pair of symmetry-related molecules which exhibit the most extensive crystal packing interactions. A stretch of single stranded RNA can be modeled with electrostatic complementarity into the interdomain cleft and continuously through the channel. These observations suggest that some form of this dimer is likely to be the functional form that unwinds double stranded RNA processively by passing one strand of RNA through the channel and passing the other strand outside of the dimer. A “descending molecular see-saw” model is proposed that is consistent with directionality of unwinding and other physicochemical properties of RNA helicases.

Bacterial Delta 5-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been intensively studied as a prototype for understanding an enzyme-catalyzed allylic rearrangement involving intramolecular proton transfer. Asp38 serves as a general base to abstract the proton from the steroid C4-H, which is a much stronger base than the carboxyl group of this residue. This unfavorable proton transfer requires 11 kcal/mol of energy which has to be provided by favorable interactions between catalytic residues and substrate in the course of the catalytic reaction. How this energy is provided at the active site of KSI has been a controversial issue, and inevitably the enzyme mechanism is not settled. To resolve these issues, we have determined the crystal structure of this enzyme at 2.3 A resolution. The crystal structure revealed that the active site environment of P. testosteroni KSI is nearly identical to that of Pseudomonas putida KSI, whose structure in complex with a reaction intermediate analogue we have determined recently. Comparison of the two structures clearly indicates that the two KSIs should share the same enzyme mechanism involving the stabilization of the dienolate intermediate by the two direct hydrogen bonds to the dienolate oxyanion, one from Tyr14 OH and the other from Asp99 COOH. Mutational analysis of the two residues and other biochemical data strongly suggest that the hydrogen bond of Tyr14 provides the more significant contribution than that of Asp99 to the requisite 11 kcal/mol of energy for the catalytic power of KSI.

A thermostable cyclodextrin glucanotransferase (CGTase) was isolated from a Bacillus stearothermophilus strain, ET1, which was screened from Korean soil. The corresponding CGTase gene cloned in Esc...

A human tumor necrosis factor-alpha (TNF-alpha) mutant (M3S) with low systemic toxicity in vivo was designed, and its structures in two different crystal packings were determined crystallographically at 1.8 and 2.15-A resolution, respectively, to explain altered biological activities of the mutant. M3S contains four changes: a hydrophilic substitution of L29S, two hydrophobic substitutions of S52I and Y56F, and a deletion of the N-terminal seven amino acids that is disordered in the structure of wild-type TNF-alpha. Compared with wild-type TNF-alpha, it exhibits 11- and 71-fold lower binding affinities for the human TNF-R55 and TNF-R75 receptors, respectively, and in vitro cytotoxic effect and in vivo systemic toxicity of M3S are 20 and 10 times lower, respectively. However, in a transplanted solid tumor mouse model, M3S suppresses tumor growth more efficiently than wild-type TNF-alpha. M3S is highly resistant to proteolysis by trypsin, and it exhibits increased thermal stability and a prolonged half-life in vivo. The L29S mutation causes substantial restructuring of the loop containing residues 29-36 into a rigid segment as a consequence of induced formation of intra- and intersubunit interactions, explaining the altered receptor binding affinity and thermal stability. A mass spectrometric analysis identified major proteolytic cleavage sites located on this loop, and thus the increased resistance of M3S to the proteolysis is consistent with the increased rigidity of the loop. The S52I and Y56F mutations do not induce a noticeable conformational change. The side chain of Phe56 projects into a hydrophobic cavity, while Ile52 is exposed to the bulk solvent. Ile52 should be involved in hydrophobic interactions with the receptors, since a mutant containing the same mutations as in M3S except for the L29S mutation exhibits an increased receptor binding affinity. The low systemic toxicity of M3S appears to be the effect of the reduced and selective binding affinities for the TNF receptors, and the superior tumor-suppression of M3S appears to be the effect of its weak but longer antitumoral activity in vivo compared with wild-type TNF-alpha. It is also expected that the 1.8-A resolution structure will serve as an accurate model for explaining the structure-function relationship of wild-type TNF-alpha and many TNF-alpha mutants reported previously and for the design of new TNF-alpha mutants.

Bacterial Δ5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testos...

The cullin–RING ubiquitin ligases are multisubunit complexes that ubiquitinate various proteins. Six different cullins encoded by the human genome selectively pair with different adaptors and substrate receptors. It is presently poorly understood how cullin-2 (Cul2) and cullin-5 (Cul5) associate specifically with their adaptor elongin BC and a SOCS-box-containing substrate receptor. Here, crystallographic and mutational analyses of a quaternary complex between the N-­terminal half of Cul5, elongin BC and SOCS2 are reported. Cul5 interacts extensively with elongin BC via residues that are highly conserved in Cul2 but not in other cullins. Cul5 also interacts with SOCS2, but via only two residues, Pro184 and Arg186, which are located in the C-terminal part of the SOCS box called the Cul5 box. Pro184 makes a ring-to-ring interaction with Trp53 of Cul5, which is substituted by alanine in Cul2. This interaction is shown to contribute significantly to the overall binding affinity between Cul5 and SOCS2–elongin BC. This study provides structural bases underlying the specificity of Cul5 and Cul2 for elongin BC and their preferential association with Cul5 or Cul2 box-containing substrate receptors.

The periplasmic histidine-binding protein, HisJ, is a receptor for the histidine permease of Salmonella typhimurium. Receptors of this type are composed of two lobes that are far apart in the unliganded structure (open conformation) and drawn close together in the liganded structure (closed conformation). The binding of the ligand, in a cleft between the lobes, stabilizes the closed conformation. Such receptors have several functions in transport: interaction with the membrane-bound complex, transmission of a transmembrane signal to hydrolyze ATP, and receiving a signal to open the lobes and release the ligand. In this study the mechanism of action of HisJ was further investigated using mutant proteins defective in ligand binding activity and closed form-specific monoclonal antibodies (Wolf, A., Shaw, E. W., Nikaido, K., and Ames G. F.-L.(1994) J. Biol. Chem. 269, 23051-23058). Y14H is defective in stabilization of the closed form, does not assume the closed empty form, and assumes an altered closed liganded form. T121A and G119R are similar to Y14H, but assume a normal closed liganded form. S72P binds the ligand to the open form, but does not assume a recognizable closed form. S92F is defective in the ability to undergo conformational change and to stabilize the closed form. All other mutant proteins appear to fall within one of these four categories. The biochemical characterization of these mutant proteins agrees with the structural analysis of the protein. We suggest that mutant proteins that do not assume the normal closed form, in addition to their defect in ligand binding, fail to interact with the membrane-bound complex and/or to transmit transmembrane signals.

The heterodimeric Rag GTPases consisting of RagA (or RagB) and RagC (or RagD) are the key regulator activating the target of rapamycin complex 1 (TORC1) in response to the level of amino acids. The heterodimer between GTP-loaded RagA/B and GDP-loaded RagC/D is the most active form that binds Raptor and leads to the activation of TORC1. Here, we present the crystal structure of Gtr1p(GTP)-Gtr2p(GDP), the active yeast Rag GTPase heterodimer. The structure reveals that GTP-to-GDP conversion on Gtr2p results in a large conformational transition of this subunit, including a large scale rearrangement of a long segment whose corresponding region in RagA is involved in binding to Raptor. In addition, the two GTPase domains of the heterodimer are brought to contact with each other, but without causing any conformational change of the Gtr1p subunit. These features explain how the nucleotide-bound statuses of the two GTPases subunits switch the Raptor binding affinity on and off.

Upon phagocytosis, Legionella pneumophila translocates numerous effector proteins into host cells to perturb cellular metabolism and immunity, ultimately establishing intracellular survival and growth. VipD of L. pneumophila belongs to a family of bacterial effectors that contain the N-terminal lipase domain and the C-terminal domain with an unknown function. We report the crystal structure of VipD and show that its C-terminal domain robustly interferes with endosomal trafficking through tight and selective interactions with Rab5 and Rab22. This domain, which is not significantly similar to any known protein structure, potently interacts with the GTP-bound active form of the two Rabs by recognizing a hydrophobic triad conserved in Rabs. These interactions prevent Rab5 and Rab22 from binding to downstream effectors Rabaptin-5, Rabenosyn-5 and EEA1, consequently blocking endosomal trafficking and subsequent lysosomal degradation of endocytic materials in macrophage cells. Together, this work reveals endosomal trafficking as a target of L. pneumophila and delineates the underlying molecular mechanism., Author Summary Legionella pneumophila is a pathogen bacterium that causes Legionnaires' disease accompanied by severe pneumonia. Surprisingly, this pathogen invades and replicates inside macrophages, whose major function is to detect and destroy invading microorganisms. How L. pneumophila can be “immune” to this primary immune cell has been a focus of intensive research. Upon being engulfed by a macrophage cell, L. pneumophila translocates hundreds of bacterial proteins into this host cell. These proteins, called bacterial effectors, are thought to manipulate normal host cellular processes. However, which host molecules and how they are targeted by the bacterial effectors are largely unknown. In this study, we determined the three-dimensional structure of L. pneumophila effector protein VipD, whose function in macrophage was unknown. Ensuing analyses revealed that VipD selectively and tightly binds two host signaling proteins Rab5 and Rab22, which are key regulators of early endosomal vesicle trafficking. These interactions prevent the activated form of Rab5 and Rab22 from binding their downstream signaling proteins, resulting in the blockade of endosomal trafficking in macrophages. The presented work shows that L. pneumophila targets endosomal Rab proteins and delineates the underlying molecular mechanism, providing a new insight into the pathogen's strategies to dysregulate normal intracellular processes.

Post-translational modification by small ubiquitin-like modifier (SUMO) can be reversed by sentrin/SUMO-specific proteases (SENPs), the first known class of deSUMOylase. Recently, we identified a new deSUMOylating enzyme DeSI-1, which is distinct from SENPs and belongs to the putative deubiquitinating isopeptidase PPPDE superfamily. Herein, we report the crystal structure of DeSI-1, revealing that this enzyme forms a homodimer and that the groove between the two subunits is the active site harboring two absolutely conserved cysteine and histidine residues that form a catalytic dyad. We also show that DeSI-1 exhibits an extremely low endopeptidase activity toward precursor forms of SUMO-1 and SUMO-2, unlike SENPs.

The substrate-binding site of a protein with multiple specificity should satisfy geometric and energetic complementarity for several different substrates. The structural basis of the multiple ligand specificity of the periplasmic lysine-, arginine-, ornithine-binding protein (LAO) was investigated by determining and analyzing the structures of the protein unliganded and liganded with each of the three high-affinity ligands (L-lysine, L-arginine, and L-ornithine) and with one low-affinity ligand (L-histidine). The geometric complementarity is achieved primarily by virtue of the large size of the ligand-binding site which can accommodate the maximum common volume of the four ligands plus three water molecules. The optimization of energetic complementarity is achieved by the relocation of protein-bound water molecules and by the movement of the Asp-11 side chain. The structure of the LAO-histidine complex indicates that the 30-fold reduced affinity of the protein for histidine is primarily due to unavailability of one ionic interaction of the histidine side chain with the protein which is present in the other three complexes.