Crystal Structure of the GTPase-activating Protein-related Domain from IQGAP1

IQGAP1 is a 190-kDa molecular scaffold containing several domains required for interaction with numerous proteins. One domain is homologous to Ras GTPase-activating protein (GAP) domains. However, instead of accelerating hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD) binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here the crystal structure of the isolated IQGAP1 GRD. Despite low sequence conservation, the overall structure of the GRD is very similar to the GAP domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the catalytic “arginine finger” seen in functional Ras GAPs, the GRD has a conserved threonine residue. GRD residues 1099–1129 have no structural equivalent in RasGAP and are seen to form an extension at one end of the molecule. Because the sequence of these residues is highly conserved, this region likely confers a functionality particular to IQGAP family GRDs. We have used isothermal titration calorimetry to demonstrate that the isolated GRD binds to active Cdc42. Assuming a mode of interaction similar to that displayed in the Ras-RasGAP complex, we created an energy-minimized model of Cdc42·GTP bound to the GRD. Residues of the GRD that contact Cdc42 map to the surface of the GRD that displays the highest level of sequence conservation. The model indicates that steric clash between threonine 1046 with the phosphate-binding loop and other subtle changes would likely disrupt the proper geometry required for GTP hydrolysis.The small GTPase Ras functions as a binary switch in cell signaling processes. When bound to GTP, Ras is able to interact with effector proteins, including Raf kinase, and alter their activities. Ras signaling is terminated when bound GTP is hydrolyzed to GDP and inorganic phosphate. The basal rate of GTP hydrolysis on Ras is quite slow (∼1.2 × 10^–4 s^–1), but this rate of hydrolysis can be enhanced ∼10⁵-fold by interaction with a GTPase-activating protein (GAP)² (1). Several RasGAPs have been identified to date including p120 RasGAP and neurofibromin (NF1). The Rho family of Ras-related small GTPases also function as binary switches in cell signaling processes. Whereas the intrinsic rate of GTP hydrolysis on Rho proteins is faster than Ras, this rate can also be stimulated by interaction with a RhoGAP. Examination of the structures of the GAP domains of p120RasGAP (2), neurofibromin (3), SynGAP (4), and the GAP domains from the RhoGAPs p50 RhoGAP and the Bcr homology domain of phosphatidylinositol 3-kinase (5, 6) indicates that although ostensibly different, these all-helical domains are structurally related (7).IQGAP1 was discovered by chance during an attempt to isolate novel matrix metalloproteinases (8). Analysis reveals that the protein contains several discrete domains and motifs including a region containing four isoleucine- and glutamine-rich motifs (IQ repeats) and a region with sequence homology to the Ras-specific GAP domains of p120RasGAP, NF1, and SynGAP (2–4, 8). Subsequently, two homologs, IQGAP2 and IQGAP3, have been discovered. The IQ repeats have been shown to mediate binding to calmodulin and calmodulin-like proteins (e.g. S100, myosin essential light chain), whereas the GAP-related domain (GRD) does not appear to bind to Ras but instead is necessary for binding to the Rho family GTPases Cdc42 and Rac1, primarily in their active forms (9–11). However, instead of accelerating hydrolysis of GTP, IQGAP1 preserves the activated states of Cdc42 and Rac1 to the extent that overexpression of IQGAP1 in cells increases the levels of active GTPase (12). Because IQGAP1 expression increases the level of activated Cdc42, initially there was some confusion as to whether the protein might not represent a novel guanine nucleotide exchange factor. However it now appears that IQGAP1 is an effector of Cdc42 and Rac1 and preserves their activated states by tightly binding to the GTPases and stabilizing them in a conformation not conducive to GTP hydrolysis. IQGAP1 appears to be such an important effector for Cdc42 that abrogation of binding to IQGAP1 not only reduces the levels of active Cdc42, it also reduces membrane-localized Cdc42 and the cellular response to bradykinin (12).A growing body of evidence implicates IQGAP1 in carcinogenesis. Expression of IQGAP1 increases during the transition from a minimally to a highly metastastic form of melanoma, and IQGAP1 has been found to be overexpressed in ovarian, breast, lung, and colorectal cancers (13–17). In vitro, overexpressed IQGAP1 enhances cell motility and invasiveness in a process that requires Cdc42 and Rac (18). β-Catenin is one of the many binding partners of IQGAP1 identified to date. IQGAP1 has been shown to bind to β-catenin and interfere with β-catenin binding to α-catenin, an interaction necessary for stable cell-cell adhesion (19). Another study found that IQGAP2 knock-out mice overexpress IQGAP1 and developage-dependent liver cancer and apoptosis (20).To better understand how a protein domain homologous to others that accelerate GTP hydrolysis can function as an effector and preserve the GTP-bound state, we have determined the x-ray structure of the IQGAP1 GRD. Despite low sequence identity, the GRD structure is quite similar to the GAP domains of p120, neurofibromin, and SynGAP; however, unlike those domains, the GRD possesses a conserved threonine in place of the catalytic arginine finger and has a 31-residue insertion that projects from one end of the molecule. Using the coordinates of Ras·GDP·AlF₃ in complex with the GAP domain of p120, we built a model of Cdc42·GTP bound to the GRD. The model indicates that a steric clash between the conserved Thr¹⁰⁴⁶ and the phosphate-binding loop of Cdc42 and other subtle changes within the active site would likely preclude nucleotide hydrolysis. Sequence conservation mapped to the surface of the GRD indicates that the surface with the highest degree of conservation overlaps with the surface that makes contacts to Cdc42 in the model.     

The Claudin Megatrachea Protein Complex

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.     

Structure of the Tyrosine-sulfated C5a Receptor N Terminus in Complex with Chemotaxis Inhibitory Protein of Staphylococcus aureus

Complement component C5a is a potent pro-inflammatory agent inducing chemotaxis of leukocytes toward sites of infection and injury. C5a mediates its effects via its G protein-coupled C5a receptor (C5aR). Although under normal conditions highly beneficial, excessive levels of C5a can be deleterious to the host and have been related to numerous inflammatory diseases. A natural inhibitor of the C5aR is chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS). CHIPS is a 121-residue protein excreted by S. aureus. It binds the N terminus of the C5aR (residues 1-35) with nanomolar affinity and thereby potently inhibits C5a-mediated responses in human leukocytes. Therefore, CHIPS provides a starting point for the development of new anti-inflammatory agents. Two O-sulfated tyrosine residues located at positions 11 and 14 within the C5aR N terminus play a critical role in recognition of C5a, but their role in CHIPS binding has not been established so far. By isothermal titration calorimetry, using synthetic Tyr-11- and Tyr-14-sulfated and non-sulfated C5aR N-terminal peptides, we demonstrate that the sulfate groups are essential for tight binding between the C5aR and CHIPS. In addition, the NMR structure of the complex of CHIPS and a sulfated C5aR N-terminal peptide reveals the precise binding motif as well as the distinct roles of sulfated tyrosine residues sY11 and sY14. These results provide a molecular framework for the design of novel CHIPS-based C5aR inhibitors.The human complement system is a key component of the innate host defense directed against invading pathogens. Complement component C5a is a 74-residue glycoprotein generated via complement activation by cleavage of the α-chain of its precursor C5. C5a is a strong chemoattractant involved in the recruitment of neutrophils and monocytes, activation of phagocytes, release of granule-based enzymes, and in the generation of oxidants (1, 2). C5a exerts its effect by activating the C5a receptor (C5aR).³ Although this is a highly efficient process, excessive or erroneous activation of the C5aR can have deleterious effects on host tissues. C5a has been implicated in the pathogenesis of many inflammatory and immunological diseases, including rheumatoid arthritis, inflammatory bowel disease, immune complex disease, and reperfusion injury (3, 4). Consequently, there is an active ongoing search for compounds that suppress C5a-mediated inflammatory responses.Chemotaxis inhibitory protein of Staphylococcus aureus (CHIPS) is a 121-residue protein excreted by S. aureus, which efficiently inhibits the activation of neutrophils and monocytes by formylated peptides and C5a (5, 6). CHIPS specifically binds to the formylated peptide receptor (FPR) and the C5aR with nanomolar affinity (K_d = 35.4 ± 7.7 nm and 1.1 ± 0.2 nm, respectively) (7), thereby suppressing the inflammatory response of the host. A CHIPS fragment lacking residues 1-30 (designated CHIPS_31-121) has the same activity in blocking the C5aR compared with wild-type CHIPS (8). CHIPS_31-121 is a compact protein comprising an α-helix packed onto a four-stranded anti-parallel β-sheet (8). C5a has an entirely different fold (PDB ID code 1KJS) and is comprised of an anti-parallel bundle of four α-helices stabilized by three disulfide bonds (9, 10). Preliminary experiments indicated that CHIPS binds exclusively to the extracellular N-terminal portion of the C5aR (7). In contrast, the binding of C5a by its receptor involves two separate binding sites: C5a residues located in the region between 12-46 (11, 12) bind to a primary binding site partly coinciding with the binding site of CHIPS, while the C terminus of C5a (residues 69-74) binds to the activation domain of the C5aR located in the receptor core (13). Because of their dissimilarity in sequence and structure, the binding sites of CHIPS and C5a are not identical (11). The present working model is that CHIPS interferes with the primary binding site of C5a located at the N terminus of the C5aR, thereby preventing the C-terminal tail of C5a from contacting the activation domain of the C5aR and blocking downstream signaling. Currently, the development of C5aR inhibitors has been focused primarily on mimicking C5a in order to directly interrupt C5a-mediated C5aR signaling (3, 4, 14). Understanding the interactions between CHIPS and the C5aR may provide valuable insights toward the development of new C5aR antagonists.Postma et al. (15) proposed that residues involved in CHIPS binding are located between residues 10-18 of the C5aR. Specifically, the acidic residues Asp-10, Asp-15, and Asp-18 and residue Gly-12 appear to be critical for binding. High affinity binding was observed between ¹²⁵I-labeled CHIPS and the N-terminal portion of the C5aR (residues 1-38) expressed on the cell surface of HEK293 cells (K_d = 29.7 ± 4.4 nm). In contrast, very moderate affinity between CHIPS and a synthetic C5aR N-terminal peptide (residues 1-37; K_d = 40 ± 19 μm), measured by isothermal titration calorimetry (ITC), was recently reported by Wright et al. (16). The discrepancy in the magnitude of these dissociation constants may be explained by the presence of two sulfate groups on tyrosine 11 and 14 of the C5aR N terminus expressed on the cell surface of HEK293 cells, which are absent in the synthetic C5aR peptide utilized by Wright et al. (16). Farzan et al. (17) stressed the critical role of these sulfate groups in activation of the C5aR by C5a. Previous mutational studies employing FITC-labeled CHIPS, however, suggested that the sulfate groups had only a limited effect on the binding affinity (15).To resolve these discrepancies, we set out to chemically synthesize several sulfated and unsulfated peptides representing the N terminus of the human C5aR. We have measured the binding affinities of these peptides to CHIPS_31-121 by ITC and used the C5aR peptide with the highest affinity to determine the structure of the complex between CHIPS_31-121 and the C5aR N terminus by NMR spectroscopy.     

Crystal Structure of the ATPase Domain of the Human AAA+ Protein Paraplegin/SPG7

Paraplegin is an m-AAA protease of the mitochondrial inner membrane that is linked to hereditary spastic paraplegias. The gene encodes an FtsH-homology protease domain in tandem with an AAA+ homology ATPase domain. The protein is believed to form a hexamer that uses ATPase-driven conformational changes in its AAA-domain to deliver substrate peptides to its protease domain. We present the crystal structure of the AAA-domain of human paraplegin bound to ADP at 2.2 Å. This enables assignment of the roles of specific side chains within the catalytic cycle, and provides the structural basis for understanding the mechanism of disease mutations.

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Reciprocal Intramolecular Interactions of Tomosyn Control Its Inhibitory Activity on SNARE Complex Formation

Neurotransmitter release from presynaptic nerve terminals is regulated by SNARE complex-mediated synaptic vesicle fusion. Tomosyn, a negative regulator of neurotransmitter release, which is composed of N-terminal WD40 repeats, a tail domain, and a C-terminal VAMP-like domain, is known to inhibit SNARE complex formation by sequestering target SNAREs (t-SNAREs) upon interaction of its C-terminal VAMP-like domain with t-SNAREs. However, it remains unclear how the inhibitory activity of tomosyn is regulated. Here we show that the tail domain functions as a regulator of the inhibitory activity of tomosyn through intramolecular interactions. The binding of the tail domain to the C-terminal VAMP-like domain interfered with the interaction of the C-terminal VAMP-like domain with t-SNAREs, and thereby repressed the inhibitory activity of tomosyn on the SNARE complex formation. The repressed inhibitory activity of tomosyn was restored by the binding of the tail domain to the N-terminal WD40 repeats. These results indicate that the probable conformational change of tomosyn mediated by the intramolecular interactions of the tail domain controls its inhibitory activity on the SNARE complex formation, leading to a regulated inhibition of neurotransmitter release.Synaptic vesicles are transported to the presynaptic plasma membrane where Ca²⁺ channels are located. Depolarization induces Ca²⁺ influx into the cytosol of nerve terminals through the Ca²⁺ channels, and this Ca²⁺ influx initiates the fusion of the vesicles with the plasma membrane, finally leading to exocytosis of neurotransmitters (1). Soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP)² receptors (SNAREs) are essential for synaptic vesicle exocytosis (2-5). Synaptic vesicles are endowed with vesicle-associated membrane protein 2 (VAMP-2) as a vesicular SNARE, whereas the presynaptic plasma membrane is endowed with syntaxin-1 and SNAP-25 as target SNAREs. VAMP-2 interacts with SNAP-25 and syntaxin-1 to form a stable SNARE complex (6-9). The formation of the SNARE complex then brings synaptic vesicles and the plasma membrane into close apposition, and provides the energy that drives the mixing of the two lipid bilayers (3-5, 9).Tomosyn is a syntaxin-1-binding protein that we originally identified (10). Tomosyn contains N-terminal WD40 repeats, a tail domain, and a C-terminal domain homologous to VAMP-2. The C-terminal VAMP-like domain (VLD) of tomosyn acts as a SNARE domain that competes with VAMP-2. Indeed, a structural study of the VLD revealed that the VLD, syntaxin-1, and SNAP-25 assemble into a SNARE complex-like structure (referred to as tomosyn complex hereafter) (11). Tomosyn inhibits SNARE complex formation by sequestering t-SNAREs through the tomosyn complex formation, and thereby inhibits SNARE-dependent neurotransmitter release. The large N-terminal region of tomosyn shares similarity to the Drosophila tumor suppressor lethal giant larvae (Lgl), the mammalian homologues M-Lgl1 and M-Lgl2, and yeast proteins Sro7p and Sro77p (12, 13). Consistent with the function of tomosyn, Lgl family members play an important role in polarized exocytosis by regulating SNARE function on the plasma membrane in yeast and epithelial cells (12, 13). However, only tomosyn, Sro7, and Sro77 have the tail domains and the VLDs, suggesting that their structural regulation is evolutionally conserved. Recently, the crystal structure of Sro7 was solved and revealed that the tail domain of Sro7 binds its WD40 repeats (14). Sec9, a yeast counterpart of SNAP-25, also binds the WD40 repeats of Sro7. This binding inhibits the SNARE complex formation and exocytosis by sequestering Sec9. In addition, binding of the tail domain to the WD40 repeats causes a conformational change of Sro7 and prevents the interaction of the WD40 repeats with Sec9, leading to regulation of the inhibitory activity of Sro7 on the SNARE complex formation (14). However, the solved structure of Sro7 lacks its VLD. Therefore, involvement of the activity of the VLD in the conformational change of Sro7 remains elusive.Genetic studies in Caenorhabditis elegans showed that TOM-1, an ortholog of vertebrate tomosyn, inhibits the priming of synaptic vesicles, and that this priming is modulated by the balance between TOM-1 and UNC-13 (15, 16). Tomosyn was also shown to be involved in inhibition of the exocytosis of dense core granules in adrenal chromaffin cells and PC12 cells (17, 18). Thus, evidence is accumulating that tomosyn acts as a negative regulator for formation of the SNARE complex, thereby inhibiting various vesicle fusion events. However, the precise molecular mechanism regulating the inhibitory action of tomosyn has yet to be elucidated.In the present study, we show that the tail domain of tomosyn binds both the WD40 repeats and the VLD and functions as a regulator for the inhibitory activity of tomosyn on the SNARE complex formation. Our results indicate that the probable conformational change of tomosyn mediated by the intramolecular interactions of the tail domain serves for controlling the inhibitory activity of the VLD.     

Crystal Structure of the N-Acetylmannosamine Kinase Domain of GNE

Background

UDP-GlcNAc 2-epimerase/ManNAc 6-kinase, GNE, is a bi-functional enzyme that plays a key role in sialic acid biosynthesis. Mutations of the GNE protein cause sialurea or autosomal recessive inclusion body myopathy/Nonaka myopathy. GNE is the only human protein that contains a kinase domain belonging to the ROK (repressor, ORF, kinase) family.

Principal Findings

We solved the structure of the GNE kinase domain in the ligand-free state. The protein exists predominantly as a dimer in solution, with small populations of monomer and higher-order oligomer in equilibrium with the dimer. Crystal packing analysis reveals the existence of a crystallographic hexamer, and that the kinase domain dimerizes through the C-lobe subdomain. Mapping of disease-related missense mutations onto the kinase domain structure revealed that the mutation sites could be classified into four different groups based on the location – dimer interface, interlobar helices, protein surface, or within other secondary structural elements.

Conclusions

The crystal structure of the kinase domain of GNE provides a structural basis for understanding disease-causing mutations and a model of hexameric wild type full length enzyme.

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Crystal Structure of the Nipah Virus Phosphoprotein Tetramerization Domain

The Nipah virus phosphoprotein (P) is multimeric and tethers the viral polymerase to the nucleocapsid. We present the crystal structure of the multimerization domain of Nipah virus P: a long, parallel, tetrameric, coiled coil with a small, α-helical cap structure. Across the paramyxoviruses, these domains share little sequence identity yet are similar in length and structural organization, suggesting a common requirement for scaffolding or spatial organization of the functions of P in the virus life cycle.     

Multiple Anaphase-promoting Complex/Cyclosome Degrons Mediate the Degradation of Human Sgo1

Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early mitosis and, thus, prevents premature sister-chromatid separation. The protein level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is down-regulated in G₁/S. Here we show that Sgo1 is degraded during the exit from mitosis, and its degradation depends on the anaphase-promoting complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C bound to Cdh1 (APC/C^Cdh1) in vitro. We have further identified two functional degradation motifs in Sgo1; that is, a KEN (Lys-Glu-Asn) box and a destruction box (D box). Although removal of either motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box deleted is stable in cells. Surprisingly, mitosis progresses normally in the presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not required for sister-chromatid separation or mitotic exit. Finally, we show that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1 steady-state protein levels in an APC/C-independent mechanism.Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs in two steps in vertebrate cells (1-3). In prophase, cohesin is phosphorylated by mitotic kinases including Plk1 and removed from chromosome arms (1, 4). Then, cleavage of centromeric cohesin by separase takes place at the metaphase-to-anaphase transition to allow sister-chromatid separation (5). The shugoshin (Sgo) family of proteins plays an important role in the protection of centromeric cohesion (6, 7). Human cells depleted of Sgo1 by RNAi undergo massive chromosome missegregation (8-11). In cells with compromised Sgo1 function, centromeric cohesin is improperly phosphorylated and removed (4, 11), resulting in premature sister-chromatid separation. It has been shown recently that Sgo1 collaborates with PP2A to counteract the action of Plk1 and other mitotic kinases and to protect centromeric cohesin from premature removal (12-14). In addition, Sgo1 has also been shown to promote stable kinetochore-microtubule attachment and sense tension across sister kinetochores (8, 15). Thus, Sgo1 is crucial for mitotic progression and chromosome segregation.Orderly progression through mitosis is regulated by the anaphase-promoting complex/cyclosome (APC/C),² a large multiprotein ubiquitin ligase that targets key mitotic regulators for destruction by the proteasome (16). APC/C selects substrates for ubiquitination by using the Cdc20 or Cdh1 activator proteins to recognize specific sequences called APC/C degrons within target proteins (17). Several APC/C degrons have been characterized, including the destruction box (D box) and the Lys-Glu-Asn box (KEN box) (18, 19). The D box, with the consensus amino acid sequence of RXXLXXXN(X indicates any amino acid), are found in many APC/C substrates, including mitotic cyclins and are essential for their ubiquitin-mediated destruction. The KEN box, which contains a consensus KEN motif, is also found in several APC/C substrates and is preferentially but not exclusively recognized by APC/C^Cdh1. When APC/C is active, it directs progression through and exit from mitosis by catalyzing the ubiquitination and timely destruction of mitotic regulators, including cyclin A, cyclin B, and the separase inhibitor securin (16). The APC/C activity needs to be tightly controlled to prevent unscheduled substrate degradation. An important mechanism for APC/C regulation is the spindle checkpoint, which prevents the activation of APC/C and destruction of its substrates in response to kinetochores that have not properly attached to the mitotic spindle (20).Recent evidence shows that Sgo1 is a substrate of APC/C, and its protein levels oscillate during the cell cycle (8, 9). In this article we study the degradation of Sgo1 in human cells. We show that Sgo1 is degraded during mitotic exit, and this degradation depends on APC/C^Cdh1. We further show that both KEN and D boxes are required for Sgo1 degradation in vivo and ubiquitination in vitro. Removal of these motifs stabilizes Sgo1 in vivo. The prolonged presence of stable Sgo1 protein in human cells does not change the kinetics of chromosome segregation and mitotic exit. Therefore, a timely scheduled degradation of Sgo1 takes place but is not required for mitotic exit. Finally, we show that Bub1 regulates Sgo1 protein levels through a mechanism that does not involve APC/C-mediated degradation.     

Crystal Structure of Cruxrhodopsin-3 from Haloarcula vallismortis

Cruxrhodopsin-3 (cR3), a retinylidene protein found in the claret membrane of Haloarcula vallismortis, functions as a light-driven proton pump. In this study, the membrane fusion method was applied to crystallize cR3 into a crystal belonging to space group P321. Diffraction data at 2.1 Å resolution show that cR3 forms a trimeric assembly with bacterioruberin bound to the crevice between neighboring subunits. Although the structure of the proton-release pathway is conserved among proton-pumping archaeal rhodopsins, cR3 possesses the following peculiar structural features: 1) The DE loop is long enough to interact with a neighboring subunit, strengthening the trimeric assembly; 2) Three positive charges are distributed at the cytoplasmic end of helix F, affecting the higher order structure of cR3; 3) The cytoplasmic vicinity of retinal is more rigid in cR3 than in bacteriorhodopsin, affecting the early reaction step in the proton-pumping cycle; 4) the cytoplasmic part of helix E is greatly bent, influencing the proton uptake process. Meanwhile, it was observed that the photobleaching of retinal, which scarcely occurred in the membrane state, became significant when the trimeric assembly of cR3 was dissociated into monomers in the presence of an excess amount of detergent. On the basis of these observations, we discuss structural factors affecting the photostabilities of ion-pumping rhodopsins.     

Crystal Structure of the Pneumococcal Vancomycin-Resistance Response Regulator DNA-Binding Domain

Vancomycin response regulator (VncR) is a pneumococcal response regulator of the VncRS two-component signal transduction system (TCS) of Streptococcus pneumoniae. VncRS regulates bacterial autolysis and vancomycin resistance. VncR contains two different functional domains, the N-terminal receiver domain and C-terminal effector domain. Here, we investigated VncR C-terminal DNA binding domain (VncRc) structure using a crystallization approach. Crystallization was performed using the micro-batch method. The crystals diffracted to a 1.964 _Å resolution and belonged to space group P212121. The crystal unit-cell parameters were a = 25.71 _Å, b = 52.97 _Å, and c = 60.61 _Å. The structure of VncRc had a helix-turn-helix motif highly similar to the response regulator PhoB of Escherichia coli. In isothermal titration calorimetry and size exclusion chromatography results, VncR formed a complex with VncS, a sensor histidine kinase of pneumococcal TCS. Determination of VncR structure will provide insight into the mechanism by how VncR binds to target genes.