J. Sivaraman

Berberine targets assembly of Escherichia coli cell division protein FtsZ.

The ever increasing problem of antibiotic resistance necessitates a search for new drug molecules that would target novel proteins in the prokaryotic system. FtsZ is one such target protein involved in the bacterial cell division machinery. In this study, we have shown that berberine, a natural plant alkaloid, targets Escherichia coli FtsZ, inhibits the assembly kinetics of the Z-ring, and perturbs cytokinesis. It also destabilizes FtsZ protofilaments and inhibits the FtsZ GTPase activity. Saturation transfer difference NMR spectroscopy of the FtsZ-berberine complex revealed that the dimethoxy groups, isoquinoline nucleus, and benzodioxolo ring of berberine are intimately involved in the interaction with FtsZ. Berberine perturbs the Z-ring morphology by disturbing its typical midcell localization and reduces the frequency of Z-rings per unit cell length to half. Berberine binds FtsZ with high affinity ( K D approximately 0.023 microM) and displaces bis-ANS, suggesting that it may bind FtsZ in a hydrophobic pocket. Isothermal titration calorimetry suggests that the FtsZ-berberine interaction occurs spontaneously and is enthalpy/entropy-driven. In silico molecular modeling suggests that the rearrangement of the side chains of the hydrophobic residues in the GTP binding pocket may facilitate the binding of the berberine to FtsZ and lead to inhibition of the association between FtsZ monomers. Together, these results clearly indicate the inhibitory role of berberine on the assembly function of FtsZ, establishing it as a novel FtsZ inhibitor that halts the first stage in bacterial cell division.

Linkers in the structural biology of protein–protein interactions

Linkers or spacers are short amino acid sequences created in nature to separate multiple domains in a single protein. Most of them are rigid and function to prohibit unwanted interactions between the discrete domains. However, Gly‐rich linkers are flexible, connecting various domains in a single protein without interfering with the function of each domain. The advent of recombinant DNA technology made it possible to fuse two interacting partners with the introduction of artificial linkers. Often, independent proteins may not exist as stable or structured proteins until they interact with their binding partner, following which they gain stability and the essential structural elements. Gly‐rich linkers have been proven useful for these types of unstable interactions, particularly where the interaction is weak and transient, by creating a covalent link between the proteins to form a stable protein–protein complex. Gly‐rich linkers are also employed to form stable covalently linked dimers, and to connect two independent domains that create a ligand‐binding site or recognition sequence. The lengths of linkers vary from 2 to 31 amino acids, optimized for each condition so that the linker does not impose any constraints on the conformation or interactions of the linked partners. Various structures of covalently linked protein complexes have been described using X‐ray crystallography, nuclear magnetic resonance and cryo‐electron microscopy techniques. In this review, we evaluate several structural studies where linkers have been used to improve protein quality, to produce stable protein–protein complexes, and to obtain protein dimers.

Structural basis for the inhibition mechanism of human cystathionine gamma-lyase, an enzyme responsible for the production of H(2)S

Impairment of the formation or action of hydrogen sulfide (H2S), an endogenous gasotransmitter, is associated with various diseases, such as hypertension, diabetes mellitus, septic and hemorrhagic shock, and pancreatitis. Cystathionine β-synthase and cystathionine γ-lyase (CSE) are two pyridoxal-5′-phosphate (PLP)-dependent enzymes largely responsible for the production of H2S in mammals. Inhibition of CSE by dl-propargylglycine (PAG) has been shown to alleviate disease symptoms. Here we report crystal structures of human CSE (hCSE), in apo form, and in complex with PLP and PLP·PAG. Structural characterization, combined with biophysical and biochemical studies, provides new insights into the inhibition mechanism of hCSE-mediated production of H2S. Transition from the open form of apo-hCSE to the closed PLP-bound form reveals large conformational changes hitherto not reported. In addition, PAG binds hCSE via a unique binding mode, not observed in PAG-enzyme complexes previously. The interaction of PAG-hCSE was not predicted based on existing information from known PAG complexes. The structure of hCSE·PLP·PAG complex highlights the particular importance of Tyr114 in hCSE and the mechanism of PAG-dependent inhibition of hCSE. These results provide significant insights, which will facilitate the structure-based design of novel inhibitors of hCSE to aid in the development of therapies for diseases involving disorders of sulfur metabolism.

Dimerization of Hepatitis E Virus Capsid Protein E2s Domain Is Essential for Virus-Host Interaction

Hepatitis E virus (HEV), a non-enveloped, positive-stranded RNA virus, is transmitted in a faecal-oral manner, and causes acute liver diseases in humans. The HEV capsid is made up of capsomeres consisting of homodimers of a single structural capsid protein forming the virus shell. These dimers are believed to protrude from the viral surface and to interact with host cells to initiate infection. To date, no structural information is available for any of the HEV proteins. Here, we report for the first time the crystal structure of the HEV capsid protein domain E2s, a protruding domain, together with functional studies to illustrate that this domain forms a tight homodimer and that this dimerization is essential for HEV–host interactions. In addition, we also show that the neutralizing antibody recognition site of HEV is located on the E2s domain. Our study will aid in the development of vaccines and, subsequently, specific inhibitors for HEV.

Structural basis for the allosteric inhibitory mechanism of human kidney-type glutaminase (KGA) and its regulation by Raf-Mek-Erk signaling in cancer cell metabolism.

Besides thriving on altered glucose metabolism, cancer cells undergo glutaminolysis to meet their energy demands. As the first enzyme in catalyzing glutaminolysis, human kidney-type glutaminase isoform (KGA) is becoming an attractive target for small molecules such as BPTES [bis-2-(5 phenylacetamido-1, 2, 4-thiadiazol-2-yl) ethyl sulfide], although the regulatory mechanism of KGA remains unknown. On the basis of crystal structures, we reveal that BPTES binds to an allosteric pocket at the dimer interface of KGA, triggering a dramatic conformational change of the key loop (Glu312-Pro329) near the catalytic site and rendering it inactive. The binding mode of BPTES on the hydrophobic pocket explains its specificity to KGA. Interestingly, KGA activity in cells is stimulated by EGF, and KGA associates with all three kinase components of the Raf-1/Mek2/Erk signaling module. However, the enhanced activity is abrogated by kinase-dead, dominant negative mutants of Raf-1 (Raf-1-K375M) and Mek2 (Mek2-K101A), protein phosphatase PP2A, and Mek-inhibitor U0126, indicative of phosphorylation-dependent regulation. Furthermore, treating cells that coexpressed Mek2-K101A and KGA with suboptimal level of BPTES leads to synergistic inhibition on cell proliferation. Consequently, mutating the crucial hydrophobic residues at this key loop abrogates KGA activity and cell proliferation, despite the binding of constitutive active Mek2-S222/226D. These studies therefore offer insights into (i) allosteric inhibition of KGA by BPTES, revealing the dynamic nature of KGAs active and inhibitory sites, and (ii) cross-talk and regulation of KGA activities by EGF-mediated Raf-Mek-Erk signaling. These findings will help in the design of better inhibitors and strategies for the treatment of cancers addicted with glutamine metabolism.

Structural basis for the neutralization and genotype specificity of hepatitis E virus

Hepatitis E virus (HEV) causes acute hepatitis in humans, predominantly by contamination of food and water, and is characterized by jaundice and flu-like aches and pains. To date, no vaccines are commercially available to prevent the disease caused by HEV. Previously, we showed that a monoclonal antibody, 8C11, specifically recognizes a neutralizing conformational epitope on HEV genotype I. The antibody 8C11 blocks the virus-like particle from binding to and penetrating the host cell. Here, we report the complex crystal structure of 8C11 Fab with HEV E2s(I) domain at 1.9 Å resolution. The 8C11 epitopes on E2s(I) were identified at Asp496-Thr499, Val510-Leu514, and Asn573-Arg578. Mutations and cell-model assays identified Arg512 as the most crucial residue for 8C11 interaction with and neutralization of HEV. Interestingly, 8C11 specifically neutralizes HEV genotype I, but not the other genotypes. Because HEV type I and IV are the most abundant genotypes, to understand this specificity further we determined the structure of E2s(IV) at 1.79 Å resolution and an E2s(IV) complex with 8C11 model was generated. The comparison between the 8C11 complexes with type I and IV revealed the key residues that distinguish these two genotypes. Of particular interest, the residue at amino acid position 497 at the 8C11 epitope region of E2s is distinct among these two genotypes. Swapping this residue from one genotype to another inversed the 8C11 reactivity, demonstrating the essential role played by amino acid 497 in the genotype recognition. These studies may lead to the development of antibody-based drugs for the specific treatment against HEV.

Crystal structures of major envelope proteins VP26 and VP28 from white spot syndrome virus shed light on their evolutionary relationship

ABSTRACT White spot syndrome virus (WSSV) is a virulent pathogen known to infect various crustaceans. It has bacilliform morphology with a tail-like appendage at one end. The envelope consists of four major proteins. Envelope structural proteins play a crucial role in viral infection and are believed to be the first molecules to interact with the host. Here, we report the localization and crystal structure of major envelope proteins VP26 and VP28 from WSSV at resolutions of 2.2 and 2.0 Å, respectively. These two proteins alone account for approximately 60% of the envelope, and their structures represent the first two structural envelope proteins of WSSV. Structural comparisons among VP26, VP28, and other viral proteins reveal an evolutionary relationship between WSSV envelope proteins and structural proteins from other viruses. Both proteins adopt β-barrel architecture with a protruding N-terminal region. We have investigated the localization of VP26 and VP28 using immunoelectron microscopy. This study suggests that VP26 and VP28 are located on the outer surface of the virus and are observed as a surface protrusion in the WSSV envelope, and this is the first convincing observation for VP26. Based on our studies combined with the literature, we speculate that the predicted N-terminal transmembrane region of VP26 and VP28 may anchor on the viral envelope membrane, making the core β-barrel protrude outside the envelope, possibly to interact with the host receptor or to fuse with the host cell membrane for effective transfer of the viral infection. Furthermore, it is tempting to extend this host interaction mode to other structural viral proteins of similar structures. Our finding has the potential to extend further toward drug and vaccine development against WSSV.

Structure and Evolutionary Origin of Ca2+-Dependent Herring Type II Antifreeze Protein

In order to survive under extremely cold environments, many organisms produce antifreeze proteins (AFPs). AFPs inhibit the growth of ice crystals and protect organisms from freezing damage. Fish AFPs can be classified into five distinct types based on their structures. Here we report the structure of herring AFP (hAFP), a Ca2+-dependent fish type II AFP. It exhibits a fold similar to the C-type (Ca2+-dependent) lectins with unique ice-binding features. The 1.7 Å crystal structure of hAFP with bound Ca2+ and site-directed mutagenesis reveal an ice-binding site consisting of Thr96, Thr98 and Ca2+-coordinating residues Asp94 and Glu99, which initiate hAFP adsorption onto the [10-10] prism plane of the ice lattice. The hAFP-ice interaction is further strengthened by the bound Ca2+ through the coordination with a water molecule of the ice lattice. This Ca2+-coordinated ice-binding mechanism is distinct from previously proposed mechanisms for other AFPs. However, phylogenetic analysis suggests that all type II AFPs evolved from the common ancestor and developed different ice-binding modes. We clarify the evolutionary relationship of type II AFPs to sugar-binding lectins.

Two-component PhoB-PhoR regulatory system and ferric uptake regulator sense phosphate and iron to control virulence genes in type III and VI secretion systems of Edwardsiella tarda.

Background: Phosphate and iron depletion by the vertebrate host can trigger bacterial virulence. Results: PhoB-PhoR and Fur sense phosphate and iron, respectively, to regulate expression of T3SS and T6SS in E. tarda through EsrC and by negative cross-talk among each other. Conclusion: T3SS and T6SS are regulated differently depending on the type of environmental factor. Significance: Understanding the regulation of bacterial virulence is crucial for controlling the pathogenicity of bacteria. Inorganic phosphate (Pi) and iron are essential nutrients that are depleted by vertebrates as a protective mechanism against bacterial infection. This depletion, however, is sensed by some pathogens as a signal to turn on the expression of virulence genes. Here, we show that the PhoB-PhoR two-component system senses changes in Pi concentration, whereas the ferric uptake regulator (Fur) senses changes in iron concentration in Edwardsiella tarda PPD130/91 to regulate the expression of type III and VI secretion systems (T3SS and T6SS) through an E. tarda secretion regulator, EsrC. In sensing low Pi concentration, PhoB-PhoR autoregulates and activates the phosphate-specific transport operon, pstSCAB-phoU, by binding directly to the Pho box in the promoters of phoB and pstS. PhoB also binds with EsrC simultaneously on the promoter of an E. tarda virulence protein, evpA, to regulate directly the transcription of genes from T6SS. In addition, PhoB requires and interacts with PhoU to activate esrC and suppress fur indirectly through unidentified regulators. Fur, on the other hand, senses high iron concentration and binds directly to the Fur box in the promoter of evpP to inhibit EsrC binding to the same region. In addition, Fur suppresses transcription of phoB, pstSCAB-phoU, and esrC indirectly via unidentified regulators, suggesting negative cross-talk with the Pho regulon. Physical interactions exist between Fur and PhoU and between Fur and EsrC. Our findings suggest that T3SS and T6SS may carry out distinct roles in the pathogenicity of E. tarda by responding to different environmental factors.

Structural and functional characterization of a novel homodimeric three-finger neurotoxin from the venom of Ophiophagus hannah (King cobra)

Snake venoms are a mixture of pharmacologically active proteins and polypeptides that have led to the development of molecular probes and therapeutic agents. Here, we describe the structural and functional characterization of a novel neurotoxin, haditoxin, from the venom of Ophiophagus hannah (King cobra). Haditoxin exhibited novel pharmacology with antagonism toward muscle (αβγδ) and neuronal (α7, α3β2, and α4β2) nicotinic acetylcholine receptors (nAChRs) with highest affinity for α7-nAChRs. The high resolution (1.5 Å) crystal structure revealed haditoxin to be a homodimer, like κ-neurotoxins, which target neuronal α3β2- and α4β2-nAChRs. Interestingly however, the monomeric subunits of haditoxin were composed of a three-finger protein fold typical of curaremimetic short-chain α-neurotoxins. Biochemical studies confirmed that it existed as a non-covalent dimer species in solution. Its structural similarity to short-chain α-neurotoxins and κ-neurotoxins notwithstanding, haditoxin exhibited unique blockade of α7-nAChRs (IC50 180 nm), which is recognized by neither short-chain α-neurotoxins nor κ-neurotoxins. This is the first report of a dimeric short-chain α-neurotoxin interacting with neuronal α7-nAChRs as well as the first homodimeric three-finger toxin to interact with muscle nAChRs.