Xuejun C. Zhang

Structural Basis for the Recognition of Blood Group Trisaccharides by Norovirus

ABSTRACT Noroviruses are one of the major causes of nonbacterial gastroenteritis epidemics in humans. Recent studies on norovirus receptors show that different noroviruses recognize different human histo-blood group antigens (HBGAs), and eight receptor binding patterns of noroviruses have been identified. The P domain of the norovirus capsids is directly involved in this recognition. To determine the precise locations and receptor binding modes of HBGA carbohydrates on the viral capsids, a recombinant P protein of a GII-4 strain norovirus, VA387, was cocrystallized with synthetic type A or B trisaccharides. Based on complex crystal structures observed at a 2.0-Å resolution, we demonstrated that the receptor binding site lies at the outermost end of the P domain and forms an extensive hydrogen-bonding network with the saccharide ligand. The A and B trisaccharides display similar binding modes, and the common fucose ring plays a key role in this interaction. The extensive interface between the two protomers in a P dimer also plays a crucial role in the formation of the receptor binding interface.

Structure of the APPL1 BAR-PH domain and characterization of its interaction with Rab5.

APPL1 is an effector of the small GTPase Rab5. Together, they mediate a signal transduction pathway initiated by ligand binding to cell surface receptors. Interaction with Rab5 is confined to the amino (N)‐terminal region of APPL1. We report the crystal structures of human APPL1 N‐terminal BAR‐PH domain motif. The BAR and PH domains, together with a novel linker helix, form an integrated, crescent‐shaped, symmetrical dimer. This BAR–PH interaction is likely conserved in the class of BAR‐PH containing proteins. Biochemical analyses indicate two independent Rab‐binding sites located at the opposite ends of the dimer, where the PH domain directly interacts with Rab5 and Rab21. Besides structurally supporting the PH domain, the BAR domain also contributes to Rab binding through a small surface region in the vicinity of the PH domain. In stark contrast to the helix‐dominated, Rab‐binding domains previously reported, APPL1 PH domain employs β‐strands to interact with Rab5. On the Rab5 side, both switch regions are involved in the interaction. Thus we identified a new binding mode between PH domains and small GTPases.

Structural basis of Rab5-Rabaptin5 interaction in endocytosis

Rab5 is a small GTPase that regulates early endosome fusion. We present here the crystal structure of the Rab5 GTPase domain in complex with a GTP analog and the C-terminal domain of effector Rabaptin5. The proteins form a dyad-symmetric Rab5–Rabaptin52–Rab5 ternary complex with a parallel coiled-coil Rabaptin5 homodimer in the middle. Two Rab5 molecules bind independently to the Rabaptin5 dimer using their switch and interswitch regions. The binding does not involve the Rab complementarity-determining regions. We also present the crystal structures of two distinct forms of GDP–Rab5 complexes, both of which are incompatible with Rabaptin5 binding. One has a dislocated and disordered switch I but a virtually intact switch II, whereas the other has its β-sheet and both switch regions reorganized. Biochemical and functional analyses show that the crystallographically observed Rab5–Rabaptin5 complex also exists in solution, and disruption of this complex by mutation abrogates endosome fusion.

The crystal structure of bovine bile salt activated lipase: insights into the bile salt activation mechanism.

BACKGROUND The intestinally located pancreatic enzyme, bile salt activated lipase (BAL), possesses unique activities for digesting different kinds of lipids. It also differs from other lipases in a requirement of bile salts for activity. A structure-based explanation for these unique properties has not been reached so far due to the absence of a three-dimensional structure. RESULTS The crystal structures of bovine BAL and its complex with taurocholate have been determined at 2.8 A resolution. The overall structure of BAL belongs to the alpha/beta hydrolase fold family. Two bile salt binding sites were found in each BAL molecule within the BAL-taurocholate complex structure. One of these sites is located close to a hairpin loop near the active site. Upon the binding of taurocholate, this loop becomes less mobile and assumes a different conformation. The other bile salt binding site is located remote from the active site. In both structures, BAL forms similar dimers with the active sites facing each other. CONCLUSIONS Bile salts activate BAL by binding to a relatively short ten-residue loop near the active site, and stabilize the loop in an open conformation. Presumably, this conformational change leads to the formation of the substrate-binding site, as suggested from kinetic data. The BAL dimer observed in the crystal structure may also play a functional role under physiological conditions.

Structures of two coronavirus main proteases: implications for substrate binding and antiviral drug design.

ABSTRACT Coronaviruses (CoVs) can infect humans and multiple species of animals, causing a wide spectrum of diseases. The coronavirus main protease (Mpro), which plays a pivotal role in viral gene expression and replication through the proteolytic processing of replicase polyproteins, is an attractive target for anti-CoV drug design. In this study, the crystal structures of infectious bronchitis virus (IBV) Mpro and a severe acute respiratory syndrome CoV (SARS-CoV) Mpro mutant (H41A), in complex with an N-terminal autocleavage substrate, were individually determined to elucidate the structural flexibility and substrate binding of Mpro. A monomeric form of IBV Mpro was identified for the first time in CoV Mpro structures. A comparison of these two structures to other available Mpro structures provides new insights for the design of substrate-based inhibitors targeting CoV Mpros. Furthermore, a Michael acceptor inhibitor (named N3) was cocrystallized with IBV Mpro and was found to demonstrate in vitro inactivation of IBV Mpro and potent antiviral activity against IBV in chicken embryos. This provides a feasible animal model for designing wide-spectrum inhibitors against CoV-associated diseases. The structure-based optimization of N3 has yielded two more efficacious lead compounds, N27 and H16, with potent inhibition against SARS-CoV Mpro.

Bcl-2 Homodimerization Involves Two Distinct Binding Surfaces, a Topographic Arrangement That Provides an Effective Mechanism for Bcl-2 to Capture Activated Bax

The homo- and heterodimerization of Bcl-2 family proteins is important for transduction and integration of apoptotic signals and control of the permeability of mitochondria and endoplasmic reticulum membranes. Here we mapped the interface of the Bcl-2 homodimer in a cell-free system using site-specific photocross-linking. Bcl-2 homodimer-specific photoadducts were detected from 11 of 17 sites studied. When modeled into the structure of Bcl-2 core, the interface is composed of two distinct surfaces: an acceptor surface that includes the hydrophobic groove made by helices 2 and 8 and the loop connecting helices 4 and 5 and a donor surface that is made by helices 1-4 and the loop connecting helices 2 and 3. The two binding surfaces are on separate faces of the three-dimensional structure, explaining the formation of Bcl-2 homodimers, homo-oligomers, and Bcl-2/Bax hetero-oligomers. We show that in vitro the Bcl-2 dimer can still interact with activated Bax as a larger oligomer. However, formation of a Bax/Bcl-2 heterodimer is favored, since this interaction inhibits Bcl-2 homodimerization. Our data support a simple model mechanism by which Bcl-2 interacts with activated Bax during apoptosis in an effective manner to neutralize the proapoptotic activity of Bax.

Bax forms an oligomer via separate, yet interdependent, surfaces

Interactions of Bcl-2 family proteins regulate permeability of the mitochondrial outer membrane and apoptosis. In particular, Bax forms an oligomer that permeabilizes the membrane. To map the interface of the Bax oligomer we used Triton X-100 as a membrane surrogate and performed site-specific photocross-linking. Bax-specific adducts were formed through photo-reactive probes at multiple sites that can be grouped into two surfaces. The first surface overlaps with the BH1–3 groove formed by Bcl-2 Homology motif 1, 2, and 3; the second surface is a rear pocket located on the opposite side of the protein from the BH1–3 groove. Further cross-linking experiments using Bax BH3 peptides and mutants demonstrated that the two surfaces interact with their counterparts in neighboring proteins to form two separated interfaces and that interaction at the BH1–3 groove primes the rear pocket for further interaction. Therefore, Bax oligomerization proceeds through a series of interactions that occur at separate, yet allosterically, coupled interfaces.

Crystal structure of cytotoxin protein suilysin from Streptococcus suis

Cholesterol-dependent cytolysins (CDC) are pore forming toxins. A prototype of the CDC family members is perfringolysin O (PFO), which directly binds to the cell membrane enriched in cholesterol, causing cell lysis. However, an exception of this general observation is intermedilysin (ILY) of Streptococcus intermedius, which requires human CD59 as a receptor in addition to cholesterol for its hemolytic activity. A possible explanation of this functional difference is the conformational variation between the C-terminal domains of the two toxins, particularly in the highly conserved undecapeptide termed tryptophan rich motif. Here, we present the crystal structure of suilysin, a CDC toxin from the infectious swine pathogen Streptococcus suis. Like PFO, suilysin does not require a host receptor for hemolytic activity; yet the crystal structure of suilysin exhibits a similar conformation in the tryptophan rich motif to ILY. This observation suggests that the current view of the structure-function relationship between CDC proteins and membrane association is far from complete.

Bcl-2 and Bax Interact via the BH1-3 Groove-BH3 Motif Interface and a Novel Interface Involving the BH4 Motif

The interaction of Bcl-2 family proteins at the mitochondrial outer membrane controls membrane permeability and thereby the apoptotic program. The anti-apoptotic protein Bcl-2 binds to the pro-apoptotic protein Bax to prevent Bax homo-oligomerization required for membrane permeabilization. Here, we used site-specific photocross-linking to map the surfaces of Bax and Bcl-2 that interact in the hetero-complex formed in a Triton X-100 micelle as a membrane surrogate. Heterodimer-specific photoadducts were detected from multiple sites in Bax and Bcl-2. Many of the interaction sites are located in the Bcl-2 homology 3 (BH3) region of Bax and the BH1–3 groove of Bcl-2 that likely form the BH3-BH1–3 groove interface. However, other interaction sites form a second interface that includes helix 6 of Bax and the BH4 region of Bcl-2. Loss-of-function mutations in the BH3 region of Bax and the BH1 region of Bcl-2 disrupted the BH3-BH1–3 interface, as expected. Surprisingly the second interface was also disrupted by these mutations. Similarly, a loss-of-function mutation in the BH4 region of Bcl-2 that forms part of the second interface also disrupted both interfaces. As expected, both kinds of mutation abolished Bcl-2-mediated inhibition of Bax oligomerization in detergent micelles. Therefore, Bcl-2 binds Bax through two interdependent interfaces to inhibit the pro-apoptotic oligomerization of Bax.

Structure of human lanthionine synthetase C-like protein 1 and its interaction with Eps8 and glutathione.

Eukaryotic lanthionine synthetase C-like protein 1 (LanCL1) is homologous to prokaryotic lanthionine cyclases, yet its biochemical functions remain elusive. We report the crystal structures of human LanCL1, both free of and complexed with glutathione, revealing glutathione binding to a zinc ion at the putative active site formed by conserved GxxG motifs. We also demonstrate by in vitro affinity analysis that LanCL1 binds specifically to the SH3 domain of a signaling protein, Eps8. Importantly, expression of LanCL1 mutants defective in Eps8 interaction inhibits nerve growth factor (NGF)-induced neurite outgrowth, providing evidence for the biological significance of this novel interaction in cellular signaling and differentiation.