Irimpan I. Mathews

Structural Genomics of the Thermotoga maritima Proteome Implemented in a High-throughput Structure Determination Pipeline

Structural genomics is emerging as a principal approach to define protein structure–function relationships. To apply this approach on a genomic scale, novel methods and technologies must be developed to determine large numbers of structures. We describe the design and implementation of a high-throughput structural genomics pipeline and its application to the proteome of the thermophilic bacterium Thermotoga maritima. By using this pipeline, we successfully cloned and attempted expression of 1,376 of the predicted 1,877 genes (73%) and have identified crystallization conditions for 432 proteins, comprising 23% of the T. maritima proteome. Representative structures from TM0423 glycerol dehydrogenase and TM0449 thymidylate synthase-complementing protein are presented as examples of final outputs from the pipeline.

New paradigm for macromolecular crystallography experiments at SSRL: automated crystal screening and remote data collection

Through the combination of robust mechanized experimental hardware and a flexible control system with an intuitive user interface, SSRL researchers have screened over 200 000 biological crystals for diffraction quality in an automated fashion. Three quarters of SSRL researchers are using these data-collection tools from remote locations.

Goniometer-based femtosecond crystallography with X-ray free electron lasers

Significance The extremely short and bright X-ray pulses produced by X-ray free-electron lasers unlock new opportunities in crystallography-based structural biology research. Efficient methods to deliver crystalline material are necessary due to damage or destruction of the crystal by the X-ray pulse. Crystals for the first experiments were 5 µm or smaller in size, delivered by a liquid injector. We describe a highly automated goniometer-based approach, compatible with crystals of larger and varied sizes, and accessible at cryogenic or ambient temperatures. These methods, coupled with improvements in data-processing algorithms, have resulted in high-resolution structures, unadulterated by the effects of radiation exposure, from only 100 to 1,000 diffraction images. The emerging method of femtosecond crystallography (FX) may extend the diffraction resolution accessible from small radiation-sensitive crystals and provides a means to determine catalytically accurate structures of acutely radiation-sensitive metalloenzymes. Automated goniometer-based instrumentation developed for use at the Linac Coherent Light Source enabled efficient and flexible FX experiments to be performed on a variety of sample types. In the case of rod-shaped Cpl hydrogenase crystals, only five crystals and about 30 min of beam time were used to obtain the 125 still diffraction patterns used to produce a 1.6-Å resolution electron density map. For smaller crystals, high-density grids were used to increase sample throughput; 930 myoglobin crystals mounted at random orientation inside 32 grids were exposed, demonstrating the utility of this approach. Screening results from cryocooled crystals of β2-adrenoreceptor and an RNA polymerase II complex indicate the potential to extend the diffraction resolution obtainable from very radiation-sensitive samples beyond that possible with undulator-based synchrotron sources.

Functional Analysis of Substrate and Cofactor Complex Structures of a Thymidylate Synthase-Complementing Protein

Like thymidylate synthase (TS) in eukaryotes, the thymidylate synthase-complementing proteins (TSCPs) are mandatory for cell survival of many prokaryotes in the absence of external sources of thymidylate. Details of the mechanism of this novel family of enzymes are unknown. Here, we report the structural and functional analysis of a TSCP from Thermotoga maritima and its complexes with substrate, analogs, and cofactor. The structures presented here provide a basis for rationalizing the TSCP catalysis and reveal the possibility of the design of an inhibitor. We have identified a new helix-loop-strand FAD binding motif characteristic of the enzymes in the TSCP family. The presence of a hydrophobic core with residues conserved among the TSCP family suggests a common overall fold.

An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene.

Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase to catalyse the methylation of the uracil moiety of 2′-deoxyuridine-5′-monophosphate. All known thymidylate synthases rely on an active site residue of the enzyme to activate 2′-deoxyuridine-5′-monophosphate. This functionality has been demonstrated for classical thymidylate synthases, including human thymidylate synthase, and is instrumental in mechanism-based inhibition of these enzymes. Here we report an example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens. Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (that is, a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2′-deoxythymidine-5′-monophosphate. These observations indicate a very different chemical cascade than that of classical thymidylate synthases or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Because several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.

Enzymatic catalysis of anti-Baldwin ring closure in polyether biosynthesis

Polycyclic polyether natural products have fascinated chemists and biologists alike owing to their useful biological activity, highly complex structure and intriguing biosynthetic mechanisms. Following the original proposal for the polyepoxide origin of lasalocid and isolasalocid and the experimental determination of the origins of the oxygen and carbon atoms of both lasalocid and monensin, a unified stereochemical model for the biosynthesis of polyether ionophore antibiotics was proposed. The model was based on a cascade of nucleophilic ring closures of postulated polyepoxide substrates generated by stereospecific oxidation of all-trans polyene polyketide intermediates. Shortly thereafter, a related model was proposed for the biogenesis of marine ladder toxins, involving a series of nominally disfavoured anti-Baldwin, endo-tet epoxide-ring-opening reactions. Recently, we identified Lsd19 from the Streptomyces lasaliensis gene cluster as the epoxide hydrolase responsible for the epoxide-opening cyclization of bisepoxyprelasalocid A to form lasalocid A. Here we report the X-ray crystal structure of Lsd19 in complex with its substrate and product analogue to provide the first atomic structure—to our knowledge—of a natural enzyme capable of catalysing the disfavoured epoxide-opening cyclic ether formation. On the basis of our structural and computational studies, we propose a general mechanism for the enzymatic catalysis of polyether natural product biosynthesis.

Three-dimensional structure of a hyperthermophilic 5'-deoxy-5'-methylthioadenosine phosphorylase from Sulfolobus solfataricus.

The structure of 5′-deoxy-5′-methylthioadenosine phosphorylase from Sulfolobus solfataricus (SsMTAP) has been determined alone, as ternary complexes with sulfate plus substrates 5′-deoxy-5′-methylthioadenosine, adenosine, or guanosine, or with the noncleavable substrate analog Formycin B and as binary complexes with phosphate or sulfate alone. The structure of unliganded SsMTAP was refined at 2.5-Å resolution and the structures of the complexes were refined at resolutions ranging from 1.6 to 2.0 Å. SsMTAP is unusual both for its broad substrate specificity and for its extreme thermal stability. The hexameric structure of SsMTAP is similar to that of purine-nucleoside phosphorylase (PNP) from Escherichia coli, however, only SsMTAP accepts 5′-deoxy-5′-methylthioadenosine as a substrate. The active site of SsMTAP is similar to that of E. coli PNP with 13 of 18 nearest residues being identical. The main differences are at Thr89, which corresponds to serine in E. coliPNP, and Glu163, which corresponds to proline in E. coli PNP. In addition, a water molecule is found near the purine N-7 position in the guanosine complex of SsMTAP. Thr89 is near the 5′-position of the nucleoside and may account for the ability of SsMTAP to accept either hydrophobic or hydrophilic substituents in that position. Unlike E. coli PNP, the structures of SsMTAP reveal a substrate-induced conformational change involving Glu163. This residue is located at the interface between subunits and swings in toward the active site upon nucleoside binding. The high-resolution structures of SsMTAP suggest that the transition state is stabilized in different ways for 6-amino versus6-oxo substrates. SsMTAP has optimal activity at 120 °C and retains full activity after 2 h at 100 °C. Examination of the three-dimensional structure of SsMTAP suggests that unlike most thermophilic enzymes, disulfide linkages play a key in role in its thermal stability.

The crystal structure of human S-adenosylmethionine decarboxylase at 2.25 A resolution reveals a novel fold.

BACKGROUND S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine synthetic pathway, and a well-studied drug target. The AdoMetDC decarboxylation reaction depends upon a pyruvoyl cofactor generated via an intramolecular proenzyme self-cleavage reaction. Both the proenzyme-processing and substrate-decarboxylation reactions are allosterically enhanced by putrescine. Structural elucidation of this enzyme is necessary to fully interpret the existing mutational and inhibitor-binding data, and to suggest further experimental studies. RESULTS The structure of human AdoMetDC has been determined to 2.25 A resolution using multiwavelength anomalous diffraction (MAD) phasing methods based on 22 selenium-atom positions. The quaternary structure of the mature AdoMetDC is an (alpha beta)2 dimer, where alpha and beta represent the products of the proenzyme self-cleavage reaction. The architecture of each (alpha beta) monomer is a novel four-layer alpha/beta-sandwich fold, comprised of two antiparallel eight-stranded beta sheets flanked by several alpha and 3(10) helices. CONCLUSIONS The structure and topology of AdoMetDC display internal symmetry, suggesting that this protein may be the product of an ancient gene duplication. The positions of conserved, functionally important residues suggest the location of the active site and a possible binding site for the effector molecule putrescine.

Crystal structure of thy1, a thymidylate synthase complementing protein from Thermotoga maritima at 2.25 Å resolution

Peter Kuhn, Scott A. Lesley, Irimpan I. Mathews, Jaume M. Canaves, Linda S. Brinen, Xiaoping Dai, Ashley M. Deacon, Marc A. Elsliger, Said Eshaghi, Ross Floyd, Adam Godzik, Carina Grittini, Slawomir K. Grzechnik, Chittibabu Guda, Keith O. Hodgson, Lukasz Jaroszewski, Cathy Karlak, Heath E. Klock, Eric Koesema, John M. Kovarik, Andreas T. Kreusch, Daniel McMullan, Timothy M. McPhillips, Mark A. Miller, Mitchell Miller, Andrew Morse, Kin Moy, Jie Ouyang, Alyssa Robb, Kevin Rodrigues, Thomas L. Selby, Glen Spraggon, Raymond C. Stevens, Susan S. Taylor, Henry van den Bedem, Jeff Velasquez, Juli Vincent, Xianhong Wang, Bill West, Guenter Wolf, John Wooley, and Ian A. Wilson* The Joint Center for Structural Genomics Stanford Synchrotron Radiation Laboratory, Stanford University, Menlo Park, California The Genomics Institute of Novartis Foundation, San Diego, California The San Diego Supercomputer Center, La Jolla, California The University of California, San Diego, La Jolla, California The Scripps Research Institute, La Jolla, California

Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1.

The guanine nucleotide exchange factor (GEF) Vav1 plays an important role in T-cell activation and tumorigenesis. In the GEF superfamily, Vav1 has the ability to interact with multiple families of Rho GTPases. The structure of the Vav1 DH-PH-CRD/Rac1 complex to 2.6 A resolution reveals a unique intramolecular network of contacts between the Vav1 cysteine-rich domain (CRD) and the C-terminal helix of the Vav1 Dbl homology (DH) domain. These unique interactions stabilize the Vav1 DH domain for its intimate association with the Switch II region of Rac1 that is critical for the displacement of the guanine nucleotide. Small angle x-ray scattering (SAXS) studies support this domain arrangement for the complex in solution. Further, mutational analyses confirms that the atypical CRD is critical for maintaining both optimal guanine nucleotide exchange activity and broader specificity of Vav family GEFs. Taken together, the data outline the detailed nature of Vav1s ability to contact a range of Rho GTPases using a novel protein-protein interaction network.