Structural analyses of the GI.4 norovirus by cryo-electron microscopy and X-ray crystallography revealing binding sites for human monoclonal antibodies.

Noroviruses are major causative agents of acute nonbacterial gastroenteritis in humans. There are neither antiviral therapeutic agents nor vaccines for noroviruses at this time. To evaluate the potential usefulness of two previously isolated human monoclonal antibody fragments, CV-1A1 and CV-2F5, we first conducted a single-particle analysis to determine the cryo-electron microscopy structure of virus-like particles (VLPs) from the genogroup I genotype 4 (GI.4) Chiba strain uniformly coated with CV-1A1 fragments. The results revealed that the GI.4-specific CV-1A1 antibody bound to the P2 subdomain, in which amino acids are less conserved and variable. Interestingly, a part of the CV-1A1 intrudes into the histo-blood group antigen-binding site, suggesting that this antibody might exert neutralizing activity. Next, we determined the crystal structure of the protruding (P) domain of the capsid protein in the complex form with the CV-2F5 antibody fragment. Consistent with the cross-reactivity, the CV-2F5 bound to the P1 subdomain, which is rich in amino acids conserved among the GI strains, and moreover induced a disruption of Chiba VLPs. These results suggest that the broadly reactive CV-2F5 antibody can be used as both a universal detection reagent and an antiviral drug for GI noroviruses. IMPORTANCE: We conducted the structural analyses of the VP1 protein from the GI.4 Chiba norovirus to identify the binding sites of the previously isolated human monoclonal antibodies CV-1A1 and CV-2F5. The cryo-electron microscopy of the Chiba virus-like particles (VLPs) complexed with the Fv-clasp forms of GI.4-specific CV-1A1 revealed that this antibody binds to the highly variable P2 subdomain, suggesting that this antibody may have neutralizing ability against the GI.4 strains. X-ray crystallography revealed that the CV-2F5 antibody bound to the P1 subdomain, which is rich in conserved amino acids. This result is consistent with the ability of the CV-2F5 antibody to react with a wide variety of GI norovirus strains. It is also found that the CV-2F5 antibody caused a disruption of VLPs. Our findings, together with previous reports on the structures of VP1 proteins and VLPs, are expected to open a path for the structure-based development of antivirals and vaccines against norovirus disease.

Targeting Ras-binding domain of ELMO1 by computational nanobody design.

The control of cell movement through manipulation of cytoskeletal structure has therapeutic prospects notably in the development of novel anti-metastatic drugs. In this study, we determine the structure of Ras-binding domain (RBD) of ELMO1, a protein involved in cytoskeletal regulation, both alone and in complex with the activator RhoG and verify its targetability through computational nanobody design. Using our dock-and-design approach optimized with native-like initial pose selection, we obtain Nb01, a detectable binder from scratch in the first-round design. An affinity maturation step guided by structure-activity relationship at the interface generates 23 Nb01 sequence variants and 17 of them show enhanced binding to ELMO1-RBD and are modeled to form major spatial overlaps with RhoG. The best binder, Nb29, inhibited ELMO1-RBD/RhoG interaction. Molecular dynamics simulation of the flexibility of CDR2 and CDR3 of Nb29 reveal the design of stabilizing mutations at the CDR-framework junctions potentially confers the affinity enhancement.

Structural basis for the dual GTPase specificity of the DOCK10 guanine nucleotide exchange factor.

Dedicator of cytokinesis 10 (DOCK10), an evolutionarily conserved guanine nucleotide exchange factor (GEF) for Rho GTPases, has the unique specificity within the DOCK-D subfamily to activate both Cdc42 and Rac, but the structural bases for these activities remained unknown. Here we present the crystal structures of the catalytic DHR2 domain of mouse DOCK10, complexed with either Cdc42 or Rac1. The structures revealed that DOCK10DHR2 binds to Cdc42 or Rac1 by slightly changing the arrangement of its two catalytic lobes. DOCK10 also has a flexible binding pocket for the 56th GTPase residue, allowing a novel interaction with Trp56Rac1. The conserved residues in switch 1 of Cdc42 and Rac1 showed common interactions with the unique Lys-His sequence in the ß5/ß6 loop of DOCK10DHR2. However, the interaction of switch 1 in Rac1 was less stable than that of switch 1 in Cdc42, due to amino acid differences at positions 27 and 30. Structure-based mutagenesis identified the DOCK10 residues that determine the Cdc42/Rac1 dual specificity.

Crystal structure of human acetylcholinesterase in complex with tacrine: Implications for drug discovery.

Alzheimer's disease (AD) is one of the most common, progressive neurodegenerative disorders affecting the aged populations. Though various disease pathologies have been suggested for AD, the impairment of the cholinergic system is one of the critical factors for the disease progression. Restoration of the cholinergic transmission through acetylcholinesterase (AChE) inhibitors is a promising disease modifying therapy. Being the first marketed drug for AD, tacrine reversibly inhibits AChE and thereby slows the breakdown of the chemical messenger acetylcholine (ACh) in the brain. However, the atomic level of interactions of tacrine towards human AChE (hAChE) is unknown for years. Hence, in the current study, we report the X-ray structure of hAChE-tacrine complex at 2.85 Å resolution. The conformational heterogeneity of tacrine within the electron density was addressed with the help of molecular mechanics assisted methods and the low-energy ligand configuration is reported, which provides a mechanistic explanation for the high binding affinity of tacrine towards AChE. Additionally, structural comparison of reported hAChE structures sheds light on the conformational selection and induced fit effects of various active site residues upon binding to different ligands and provides insight for future drug design campaigns against AD where AChE is a drug target.

Conformational alterations in unidirectional ion transport of a light-driven chloride pump revealed using X-ray free electron lasers.

Light-driven chloride-pumping rhodopsins actively transport anions, including various halide ions, across cell membranes. Recent studies using time-resolved serial femtosecond crystallography (TR-SFX) have uncovered the structural changes and ion transfer mechanisms in light-driven cation-pumping rhodopsins. However, the mechanism by which the conformational changes pump an anion to achieve unidirectional ion transport, from the extracellular side to the cytoplasmic side, in anion-pumping rhodopsins remains enigmatic. We have collected TR-SFX data of Nonlabens marinus rhodopsin-3 (NM-R3), derived from a marine flavobacterium, at 10-µs and 1-ms time points after photoexcitation. Our structural analysis reveals the conformational alterations during ion transfer and after ion release. Movements of the retinal chromophore initially displace a conserved tryptophan to the cytoplasmic side of NM-R3, accompanied by a slight shift of the halide ion bound to the retinal. After ion release, the inward movements of helix C and helix G and the lateral displacements of the retinal block access to the extracellular side of NM-R3. Anomalous signal data have also been obtained from NM-R3 crystals containing iodide ions. The anomalous density maps provide insight into the halide binding site for ion transfer in NM-R3.

Structural insights into the small GTPase specificity of the DOCK guanine nucleotide exchange factors.

The dedicator of cytokinesis (DOCK) family of guanine nucleotide exchange factors (GEFs) regulates cytoskeletal dynamics by activating the GTPases Rac and/or Cdc42. Eleven human DOCK proteins play various important roles in developmental processes and the immune system. Of these, DOCK1-5 proteins bind to engulfment and cell motility (ELMO) proteins to perform their physiological functions. Recent structural studies have greatly enhanced our understanding of the complex and diverse mechanisms of DOCK GEF activity and GTPase recognition and its regulation by ELMO. This review is focused on gaining structural insights into the substrate specificity of the DOCK GEFs, and discuss how Rac and Cdc42 are specifically recognized by the catalytic DHR-2 and surrounding domains of DOCK or binding partners.

Piperidine-4-carboxamide as a new scaffold for designing secretory glutaminyl cyclase inhibitors.

Alzheimer's disease (AD), a common chronic neurodegenerative disease, has become a major public health concern. Despite years of research, therapeutics for AD are limited. Overexpression of secretory glutaminyl cyclase (sQC) in AD brain leads to the formation of a highly neurotoxic pyroglutamate variant of amyloid beta, pGlu-Aß, which acts as a potential seed for the aggregation of full length Aß. Preventing the formation of pGlu-Aß through inhibition of sQC has become an attractive disease-modifying therapy in AD. In this current study, through a pharmacophore assisted high throughput virtual screening, we report a novel sQC inhibitor (Cpd-41) with a piperidine-4-carboxamide moiety (IC50 = 34 µM). Systematic molecular docking, MD simulations and X-ray crystallographic analysis provided atomistic details of the binding of Cpd-41 in the active site of sQC. The unique mode of binding and moderate toxicity of Cpd-41 make this molecule an attractive candidate for designing high affinity sQC inhibitors.

Isoprenoid-chained lipid EROCOC17+4: a new matrix for membrane protein crystallization and a crystal delivery medium in serial femtosecond crystallography.

In meso crystallization of membrane proteins relies on the use of lipids capable of forming a lipidic cubic phase (LCP). However, almost all previous crystallization trials have used monoacylglycerols, with 1-(cis-9-octadecanoyl)-rac-glycerol (MO) being the most widely used lipid. We now report that EROCOC17+4 mixed with 10% (w/w) cholesterol (Fig. 1) serves as a new matrix for crystallization and a crystal delivery medium in the serial femtosecond crystallography of Adenosine A2A receptor (A2AR). The structures of EROCOC17+4-matrix grown A2AR crystals were determined at 2.0 Å resolution by serial synchrotron rotation crystallography at a cryogenic temperature, and at 1.8 Å by LCP-serial femtosecond crystallography, using an X-ray free-electron laser at 4 and 20 °C sample temperatures, and are comparable to the structure of the MO-matrix grown A2AR crystal (PDB ID: 4EIY). Moreover, X-ray scattering measurements indicated that the EROCOC17+4/water system did not form the crystalline LC phase at least down to - 20 °C, in marked contrast to the equilibrium MO/water system, which transforms into the crystalline LC phase below about 17 °C. As the LC phase formation within the LCP-matrix causes difficulties in protein crystallography experiments in meso, this feature of EROCOC17+4 will expand the utility of the in meso method.

Structural Basis for the Dual Substrate Specificity of DOCK7 Guanine Nucleotide Exchange Factor.

The Dedicator Of CytoKinesis (DOCK) family of atypical guanine nucleotide exchange factors activates the Rho family GTPases Rac and/or Cdc42 through DOCK homology region 2 (DHR-2). Previous structural analyses of the DHR-2 domains of DOCK2 and DOCK9 have shown that they preferentially bind Rac1 and Cdc42, respectively; however, the molecular mechanism by which DHR-2 distinguishes between these GTPases is unclear. Here we report the crystal structure of the Cdc42-bound form of the DOCK7 DHR-2 domain showing dual specificity for Rac1 and Cdc42. The structure revealed increased substrate tolerance of DOCK7 at the interfaces with switch 1 and residue 56 of Cdc42. Furthermore, molecular dynamics simulations showed a closed-to-open conformational change in the DOCK7 DHR-2 domain between the Cdc42- and Rac1-bound states by lobe B displacement. Our results suggest that lobe B acts as a sensor for identifying different switch 1 conformations and explain how DOCK7 recognizes both Rac1 and Cdc42.

Grease matrix as a versatile carrier of proteins for serial crystallography.

Serial femtosecond X-ray crystallography (SFX) has revolutionized atomic-resolution structural investigation by expanding applicability to micrometer-sized protein crystals, even at room temperature, and by enabling dynamics studies. However, reliable crystal-carrying media for SFX are lacking. Here we introduce a grease-matrix carrier for protein microcrystals and obtain the structures of lysozyme, glucose isomerase, thaumatin and fatty acid-binding protein type 3 under ambient conditions at a resolution of or finer than 2 Å.

Structures of an ATP-independent Lon-like protease and its complexes with covalent inhibitors.

The Lon proteases are a unique family of chambered proteases with a built-in AAA+ (ATPases associated with diverse cellular activities) module. Here, crystal structures of a unique member of the Lon family with no intrinsic ATPase activity in the proteolytically active form are reported both alone and in complexes with three covalent inhibitors: two peptidomimetics and one derived from a natural product. This work reveals the unique architectural features of an ATP-independent Lon that selectively degrades unfolded protein substrates. Importantly, these results provide mechanistic insights into the recognition of inhibitors and polypeptide substrates within the conserved proteolytic chamber, which may aid the development of specific Lon-protease inhibitors.

Direct metal recognition by guanine nucleotide-exchange factor in the initial step of the exchange reaction.

Rab small GTPases regulate vesicle transport in eukaryotes by interacting with various effectors. Guanine nucleotide-exchange factor (GEF) catalyzes the transition from inactive GDP-bound Rab to active GTP-bound Rab. The existence of several GDP-bound intermediates containing the Arabidopsis thaliana Rab5 homologue ARA7 and the GEF VPS9a prior to the formation of a nucleotide-free binary complex has been proposed [Uejima et al. (2010), J. Biol. Chem. 285, 36689-36697]. During this process, VPS9a directly interacts with the ß-phosphate of GDP and the P-loop lysine of ARA7 via a catalytically important aspartate finger, which promotes the release of GDP from ARA7. However, it is unclear how VPS9a removes Mg2+ from ARA7 before forming the GDP-bound ternary complex. Here, the structure of the ARA7-GDP-Ca2+-VPS9a complex is reported, in which the aspartate finger directly coordinates the divalent metal ion. Ca2+ is bound to the canonical Mg2+-binding site, coordinated by the ß-phosphate of GDP and the P-loop serine of ARA7. Unexpectedly, Ca2+ is further coordinated by the aspartate finger and the main chain of VPS9a. This structure may represent the earliest intermediate step in the GEF-catalyzed nucleotide-exchange reaction of ARA7 before the metal-free GDP-bound intermediates are created.

Structural switching of Cu,Zn-superoxide dismutases at loop VI: insights from the crystal structure of 2-mercaptoethanol-modified enzyme.

Cu,Zn SOD1 (superoxide dismutase 1) is implicated in FALS (familial amyotrophic lateral sclerosis) through the accumulation of misfolded proteins that are toxic to neuronal cells. Loop VI (residues 102-115) of the protein is at the dimer interface and could play a critical role in stability. The free cysteine residue, Cys111 in the loop, is readily oxidized and alkylated. We have found that modification of this Cys111 with 2-ME (2-mercaptoethanol; 2-ME-SOD1) stabilizes the protein and the mechanism may provide insights into destabilization and the formation of aggregated proteins. Here, we determined the crystal structure of 2-ME-SOD1 and find that the 2-ME moieties in both subunits interact asymmetrically at the dimer interface and that there is an asymmetric configuration of segment Gly108 to Cys111 in loop VI. One loop VI of the dimer forms a 310-helix (Gly108 to His110) within a unique ß-bridge stabilized by a hydrogen bond between Ser105-NH and His110-CO, while the other forms a ß-turn without the H-bond. The H-bond (H-type) and H-bond free (F-type) configurations are also seen in some wild-type and mutant human SOD1s in the Protein Data Bank suggesting that they are interconvertible and an intrinsic property of SOD1s. The two structures serve as a basis for classification of these proteins and hopefully a guide to their stability and role in pathophysiology.

GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP ß-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP ß-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

Elucidation of Rab27 recruitment by its effectors: structure of Rab27a bound to Exophilin4/Slp2-a.

Rab GTPases coordinate vesicular trafficking within eukaryotic cells by collaborating with a set of effector proteins. Rab27a regulates numerous exocytotic pathways, and its dysfunction causes the Griscelli syndrome human immunodeficiency. Exophilin4/Slp2-a localizes on phosphatidylserine-enriched plasma membrane, and its N-terminal Rab27-binding domain (RBD27) specifically recognizes Rab27 on the surfaces of melanosomes and secretory granules prior to docking and fusion. To characterize the selective binding of Rab27 to 11 various effectors, we have determined the 1.8 A resolution structure of Rab27a in complex with Exophilin4 RBD27. The effector packs against the switch and interswitch elements of Rab27a, and specific affinity toward Rab27a is modulated by a shift in the orientation of the effector structural motif (S/T)(G/L)xW(F/Y)(2). The observed structural complementation between the interacting surfaces of Rab27a and Exophilin4 sheds light on the disparities among the Rab27 effectors and outlines a general mechanism for their recruitment.

Purification, crystallization and preliminary X-ray crystallographic analysis of Rab27a GTPase in complex with exophilin4/Slp2-a effector.

By switching between GTP-active and GDP-inactive conformations, small Ras GTPases partly regulate membrane trafficking, cell growth and cytoskeleton dynamics. Among Rab GTPases, the Rab27 subfamily, which comprises Rab27a and Rab27b, controls the proper targeting of secretory vesicles to the plasma membrane. GppNHp-bound Rab27a in complex with the Rab27-binding domain of exophilin4/Slp2-a effector has been purified and crystallized for structural studies. The crystals belong to space group P2(1)2(1)2(1) and a complete data set was collected to a resolution of 1.8 A. Eventually, the structural characterization of the Rab27a-exophilin4/Slp2-a complex will clarify Rab27 recognition by its effectors prior to vesicle tethering and docking.

Structure of the small GTPase Rab27b shows an unexpected swapped dimer.

Members of the Rab family of small GTPases regulate membrane traffic within the cell by recruiting their specific effectors in a nucleotide-dependent manner. The Rab27 subfamily consists of Rab27a and Rab27b, which share 70% sequence identity. By interacting with a large set of effector proteins such as melanophilin and granuphilin, both Rab27a and Rab27b regulate the exocytosis of secretory lysosomes. Here, the crystal structures of mouse Rab27b in complex with GDP have been determined in three distinct crystal lattices. Surprisingly, Rab27b-GDP exists in an open conformation with protruding switch and interswitch regions, which are stabilized through dimerization by means of domain-swapping in the crystals. In contrast, small-angle X-ray scattering measurements showed an extended monomer form of Rab27b in solution. The observed dimer formation of Rab27b-GDP in the crystals would restrain the highly flexible switch regions. Possible biological implications of this atypical structure of Rab27b and its plausible influence in effector interaction are discussed.

Molecular basis for autoregulatory interaction between GAE domain and hinge region of GGA1.

Golgi-localizing, gamma-adaptin ear domain homology, ADP ribosylation factor-binding (GGA) proteins and the adaptor protein (AP) complex, AP-1, are involved in membrane traffic between the trans Golgi network and the endosomes. The gamma-adaptin ear (GAE) domain of GGAs and the gamma1 ear domain of AP-1 interact with an acidic phenylalanine motif found in accessory proteins. The GAE domain of GGA1 (GGA1-GAE) interacts with a WNSF-containing peptide derived from its own hinge region, although the peptide sequence deviates from the standard acidic phenylalanine motif. We report here the structure of GGA1-GAE in complex with the GGA1 hinge peptide, which revealed that the two aromatic side chains of the WNSF sequence fit into a hydrophobic groove formed by aliphatic portions of the side chains of conserved arginine and lysine residues of GGA1-GAE, in a similar manner to the interaction between GGA-GAEs and acidic phenylalanine sequences from the accessory proteins. Fluorescence quenching experiments indicate that the GGA1 hinge region binds to GGA1-GAE and competes with accessory proteins for binding. Taken together with the previous observation that gamma1 ear binds to the GGA1 hinge region, the interaction between the hinge region and the GAE domain underlies the autoregulation of GGA function in clathrin-mediated trafficking through competing with the accessory proteins and the AP-1 complex.

Development of an automated large-scale protein-crystallization and monitoring system for high-throughput protein-structure analyses.

Protein crystallization remains one of the bottlenecks in crystallographic analysis of macromolecules. An automated large-scale protein-crystallization system named PXS has been developed consisting of the following subsystems, which proceed in parallel under unified control software: dispensing precipitants and protein solutions, sealing crystallization plates, carrying robot, incubators, observation system and image-storage server. A sitting-drop crystallization plate specialized for PXS has also been designed and developed. PXS can set up 7680 drops for vapour diffusion per hour, which includes time for replenishing supplies such as disposable tips and crystallization plates. Images of the crystallization drops are automatically recorded according to a preprogrammed schedule and can be viewed by users remotely using web-based browser software. A number of protein crystals were successfully produced and several protein structures could be determined directly from crystals grown by PXS. In other cases, X-ray quality crystals were obtained by further optimization by manual screening based on the conditions found by PXS.

Structure of the ectodomain of Drosophila peptidoglycan-recognition protein LCa suggests a molecular mechanism for pattern recognition.

The peptidoglycan-recognition protein LCa (PGRP-LCa) is a transmembrane receptor required for activation of the Drosophila immune deficiency pathway by monomeric Gram-negative peptidoglycan. We have determined the crystal structure of the ectodomain of PGRP-LCa at 2.5-A resolution and found two unique helical insertions in the LCa ectodomain that disrupt an otherwise L-shaped peptidoglycan-docking groove present in all other known PGRP structures. The deficient binding of PGRP-LCa to monomeric peptidoglycan was confirmed by biochemical pull-down assays. Recognition of monomeric peptidoglycan involves both PGRP-LCa and -LCx. We showed that association of the LCa and LCx ectodomains in vitro depends on monomeric peptidoglycan. The presence of a defective peptidoglycan-docking groove, while preserving a unique role in mediating monomeric peptidoglycan induction of immune response, suggests that PGRP-LCa recognizes the exposed structural features of a monomeric muropeptide when the latter is bound to and presented by the ectodomain of PGRP-LCx. Such features include N-acetyl glucosamine and the anhydro bond in the glycan of the muropeptide, which have been demonstrated to be critical for immune stimulatory activity.