Jorg Hendle

Structural analysis of a set of proteins resulting from a bacterial genomics project

The targets of the Structural GenomiX (SGX) bacterial genomics project were proteins conserved in multiple prokaryotic organisms with no obvious sequence homolog in the Protein Data Bank of known structures. The outcome of this work was 80 structures, covering 60 unique sequences and 49 different genes. Experimental phase determination from proteins incorporating Se‐Met was carried out for 45 structures with most of the remainder solved by molecular replacement using members of the experimentally phased set as search models. An automated tool was developed to deposit these structures in the Protein Data Bank, along with the associated X‐ray diffraction data (including refined experimental phases) and experimentally confirmed sequences. BLAST comparisons of the SGX structures with structures that had appeared in the Protein Data Bank over the intervening 3.5 years since the SGX target list had been compiled identified homologs for 49 of the 60 unique sequences represented by the SGX structures. This result indicates that, for bacterial structures that are relatively easy to express, purify, and crystallize, the structural coverage of gene space is proceeding rapidly. More distant sequence‐structure relationships between the SGX and PDB structures were investigated using PDB‐BLAST and Combinatorial Extension (CE). Only one structure, SufD, has a truly unique topology compared to all folds in the PDB. Proteins 2005.

A novel mode of Gleevec binding is revealed by the structure of spleen tyrosine kinase

Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine → phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.

SGX523 is an exquisitely selective, ATP-competitive inhibitor of the MET receptor tyrosine kinase with antitumor activity in vivo

The MET receptor tyrosine kinase has emerged as an important target for the development of novel cancer therapeutics. Activation of MET by mutation or gene amplification has been linked to kidney, gastric, and lung cancers. In other cancers, such as glioblastoma, autocrine activation of MET has been demonstrated. Several classes of ATP-competitive inhibitor have been described, which inhibit MET but also other kinases. Here, we describe SGX523, a novel, ATP-competitive kinase inhibitor remarkable for its exquisite selectivity for MET. SGX523 potently inhibited MET with an IC50 of 4 nmol/L and is >1,000-fold selective versus the >200-fold selectivity of other protein kinases tested in biochemical assays. Crystallographic study revealed that SGX523 stabilizes MET in a unique inactive conformation that is inaccessible to other protein kinases, suggesting an explanation for the selectivity. SGX523 inhibited MET-mediated signaling, cell proliferation, and cell migration at nanomolar concentrations but had no effect on signaling dependent on other protein kinases, including the closely related RON, even at micromolar concentrations. SGX523 inhibition of MET in vivo was associated with the dose-dependent inhibition of growth of tumor xenografts derived from human glioblastoma and lung and gastric cancers, confirming the dependence of these tumors on MET catalytic activity. Our results show that SGX523 is the most selective inhibitor of MET catalytic activity described to date and is thus a useful tool to investigate the role of MET kinase in cancer without the confounding effects of promiscuous protein kinase inhibition. [Mol Cancer Ther 2009;8(12):3181–90]

Fragment-based discovery of hepatitis C virus NS5b RNA polymerase inhibitors.

Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with approximately 1-10mM binding affinity (K(D)) were iteratively optimized to give leads with approximately 200nM biochemical activity and low microM cellular activity in a Replicon assay.

The 1.59 Å resolution crystal structure of TM0096, a flavin mononucleotide binding protein from Thermotoga maritima

Introduction. The TM0096 gene from Thermotoga maritima is distributed widely among microorganisms, suggesting that it performs a function indispensable for life and/or virulence. Homologs of the TM0096 protein are found in prokaryotes as well as eukaryotes, including the pathogens Yersinia pestis, Listeria, Clostridium, and Bacillus anthracis. TM0096 belongs to the NIFR3 family of proteins, named after the nifR3 gene from Rhodobacter. The biochemical functions of NIFR3 family proteins are unclear, but limited research points to a role in nitrogen metabolism. For example, the photosynthetic purple bacteria Rhodobacter capsulatus experiences an order of magnitude increase in NIFR3 expression under limiting nitrogen conditions. The nifR3-ntrB-ntrC operon is translationally controlled by the nitrogen-sensing two-component regulator NTRC. Although T. maritima TM0096 shares 32% sequence identity with the R. capsulatus NIFR3 protein, operon organization is not conserved. A second example is found in Sterkiella histriomuscorum. In a stressful environment (such as limiting nutrient availability) this protist transforms into dormant encysted cells and re-transforms into vegetative cells when favorable conditions return. The dormant cells contain a pool of mRNA transcripts for a NIFR3-like protein. This report describes the structure of TM0096 at 1.59 Å resolution. The initial annotation of the TM0096 gene as a TIM-barrel “putative flavin oxidoreductase” is substantiated by the presence of a flavin mononucleotide (FMN) cofactor in the structure. Indications about the protein function gleaned from this model could be helpful in protein engineering applications or the structure-informed design of potential inhibitors.

Crystal structure of YIGZ, a conserved hypothetical protein from Escherichia coli k12 with a novel fold

Introduction. The yigZ gene from Escherichia coli K12 is widely conserved among thermophiles, archaea, and pathogens including Yersinia pestis, Vibrio cholerae and Salmonella typhimurium. While the function for this protein remains uncharacterized, domain conservation patterns suggest that the yigZ gene product plays an indispensable function, making it a possible antimicrobial drug target. The closest mammalian homologue of yigZ is the gene impact (Accession ID: AAG35736). Mouse impact is an imprinted gene but little else is known of its function. Imprinted genes are expressed in a parent-of-origindependent manner, and generally have roles in differentiation, development, and regulation of cell proliferation. Aberrations in imprinted genes or their regulation have been implicated in various human diseases including Prader-Willi syndrome, Angelman syndrome, diabetes mellitus, bipolar affective disorder, and some malignant tumors. Knowledge of the YIGZ protein structure provides a potential template to model the structure of the IMPACT protein and provide clues regarding biochemical function. This report describes the x-ray structure determination of YIGZ to 2.8 Å resolution. Structural homology provides some insights into the function of this protein and reveals a novel polypeptide chain fold.

X-ray crystallographic characterization and phasing of an NtrC homologue.

The ZraR (HydG) protein is a 441-amino-acid protein with three functional domains and is homologous to the general nitrogen-regulatory protein NtrC that regulates nitrogen assimilation in many bacteria. The AAA and DNA-binding domains (residues 141-441) of the ZraR protein from Salmonella typhimurium were crystallized using the sitting-drop vapour-diffusion method. X-ray diffraction data from the native crystal have been collected to 3.0 A resolution. Initial phasing was successfully performed by the SIRAS method using derivativatized crystals soaked in 1 mM ethylmercuric phosphate. Preliminary structural analysis shows the presence of a hexamer in the asymmetric unit. Model building is in progress.