Akil Dharamsi

Structural proteomics of an archaeon.

A set of 424 nonmembrane proteins from Methanobacterium thermoautotrophicum were cloned, expressed and purified for structural studies. Of these, ∼20% were found to be suitable candidates for X-ray crystallographic or NMR spectroscopic analysis without further optimization of conditions, providing an estimate of the number of the most accessible structural targets in the proteome. A retrospective analysis of the experimental behavior of these proteins suggested some simple relations between sequence and solubility, implying that data bases of protein properties will be useful in optimizing high throughput strategies. Of the first 10 structures determined, several provided clues to biochemical functions that were not detectable from sequence analysis, and in many cases these putative functions could be readily confirmed by biochemical methods. This demonstrates that structural proteomics is feasible and can play a central role in functional genomics.

Protein production: feeding the crystallographers and NMR spectroscopists

Protein purification efforts for structural genomics will focus on automation for the readily-expressed proteins, and process development for the more difficult ones, such as membrane proteins. Thousands of proteins are expected to be produced in the next few years. The purified proteins will be valuable reagents for the entire research community.

Structural proteomics: prospects for high throughput sample preparation

1. BackgroundWith the near completion of many genome sequencing projects has come the soberingrealisation that our understanding of biology is nowhere near complete. For example, inthe worm, C. elegans, less than half of the predicted proteins have a known function(Consortium, 1998). The major challenge facing biologists in the next decade will be to‘‘finish the job’’, that is, to ascribe a function to each of the proteins that have been discovered

Crystal structure of dTDP-4-keto-6-deoxy-D-hexulose 3,5-epimerase from Methanobacterium thermoautotrophicum complexed with dTDP.

Deoxythymidine diphosphate (dTDP)-4-keto-6-deoxy-d-hexulose 3,5-epimerase (RmlC) is involved in the biosynthesis of dTDP-l-rhamnose, which is an essential component of the bacterial cell wall. The crystal structure of RmlC from Methanobacterium thermoautotrophicumwas determined in the presence and absence of dTDP, a substrate analogue. RmlC is a homodimer comprising a central jelly roll motif, which extends in two directions into longer β-sheets. Binding of dTDP is stabilized by ionic interactions to the phosphate group and by a combination of ionic and hydrophobic interactions with the base. The active site, which is located in the center of the jelly roll, is formed by residues that are conserved in all known RmlC sequence homologues. The conservation of the active site residues suggests that the mechanism of action is also conserved and that the RmlC structure may be useful in guiding the design of antibacterial drugs.

Insights into ligand binding and catalysis of a central step in NAD+ synthesis: structures of Methanobacterium thermoautotrophicum NMN adenylyltransferase complexes.

Nicotinamide mononucleotide adenylyltransferase (NMNATase) catalyzes the linking of NMN+ or NaMN+ with ATP, which in all organisms is one of the common step in the synthesis of the ubiquitous coenzyme NAD+, via both de novo and salvage biosynthetic pathways. The structure of Methanobacterium thermoautotrophicum NMNATase determined using multiwavelength anomalous dispersion phasing revealed a nucleotide-binding fold common to nucleotidyltransferase proteins. An NAD+ molecule and a sulfate ion were bound in the active site allowing the identification of residues involved in product binding. In addition, the role of the conserved16HXGH19 active site motif in catalysis was probed by mutagenic, enzymatic and crystallographic techniques, including the characterization of an NMN+/SO 4 2 – complex of mutant H19A NMNATase.

Deep trefoil knot implicated in RNA binding found in an archaebacterial protein

The M. thermoautotrophicum MT1 gene is conserved in archaea, it lies in a ribosomal protein operon, and it codes for 268 amino acid protein of unknown function. We report here the structure of MT1 that is novel from several standpoints: (i) the structure contains a novel topological unit -- a deep C-terminal trefoil knot first observed in a TIM barrel-like fold, archaebacterial proteins and rarely observed in other proteins; (ii) structurally, it contains only five ({beta}{alpha}) units, and the arrangements of its hydrophobic and hydrophilic surfaces are opposite to that found in classical TIM barrel proteins; (iii) functionally, although it lacks typical features found in enzymes of the barrel family, it has strongly conserved residues clustered on the surface that form a potential catalytic-site; (iv), the structure provides a first example of barrel-like fold linked to an RNA-binding domain, suggesting an extension of TIM barrel functionality to nucleic acid binding and/or catalysis.

Rapid fold and structure determination of the archaeal translation elongation factor 1beta from Methanobacterium thermoautotrophicum.

The tertiary fold of the elongation factor, aEF-1β, from Methanobacterium thermoautotrophicum was determined in a high-throughput fashion using a minimal set of NMR experiments. NMR secondary structure prediction, deuterium exchange experiments and the analysis of chemical shift perturbations were combined to identify the protein fold as an alpha-beta sandwich typical of many RNA binding proteins including EF-G. Following resolution of the tertiary fold, a high resolution structure of aEF-1β was determined using heteronuclear and homonuclear NMR experiments and a semi-automated NOESY assignment strategy. Analysis of the aEF-1β structure revealed close similarity to its human analogue, eEF-1β. In agreement with studies on EF-Ts and human EF-1β, a functional mechanism for nucleotide exchange is proposed wherein Phe46 on an exposed loop acts as a lever to eject GDP from the associated elongation factor G-protein, aEF-1α. aEF-1β was also found to bind calcium in the groove between helix α2 and strand β4. This novel feature was not observed previously and may serve a structural function related to protein stability or may play a functional role in archaeal protein translation.