Book:Structural Proteomics and its Impact on the Life Sciences:6

Chapter 6: The Contribution of Structural Proteomics to Understanding the Function of Hypothetical Proteins

Michael D. Suits, Allan Matte, Zongchao Jia and Miroslaw Cygler ^[1]

Molecular Tour
Structural Genomics efforts throughout the world have led to the development of high-throughput (HTP) techniques from go from genes to 3D structures. These HTP methods have yield thousands of new structures, many of which are from hypothetical proteins, with less than 30% sequence identity to a protein of known 3D structure, and are unknown function. In order to attempt to make functional assignments for these proteins, The Montreal-Kingston Bacterial Structural Genomics Initiative [1] has been utilizing 3D structural information of these newly determined proteins. Here are illustrations of several such examples.

A quinate/shikimate dehydrogenase from Escherichia coli - YdiB

, as it possessed 28% sequence identity to E. coli AroE, a characterized shikimate dehydrogenase. (Chaudhuri S & Coggins JR 1985 The purification of shikimate dehydrogenase from Escherichia coli. Biochem J 226 217-223; Anton IA & Coggins JR 1988 Sequencing and overexpression of the Escherichia coli aroE gene encoding shikimate dehydrogenase. Biochem J 249 319-326.)

(Fig. 1) [Michel G, Roszak AW, Sauve V, Maclean J, Matte A, Coggins JR, Cygler M & Lapthorn AJ 2003 Structures of shikimate dehydrogenase AroE and its Paralog YdiB. A common structural framework for different activities. J Biol Chem 278 19463-19472]. . Both enzymes share similar folds, contain signature motifs indicative of dinucleotide (NAD+, NADP+) binding, and while AroE is a monomer, the crystal structure of E. coli YdiB revealed a dimer [Michel, 2003].

, as it involves a relatively , but is consistent with the molecular weight determined by size exclusion chromatography. Both enzymes consist of two α/β domains, with the C-terminal domain adopting a canonical Rossmann fold topology, consistent with dinucleotide binding.

Escherichia coli Proteins Associated With Heme Metabolism

At the initiation of our investigation ChuS, from Shigella dysenteriae, was known only as a protein up-regulated under low iron conditions, responsible for the prevention of heme toxicity. It showed low sequence homology to know 3D structures and thus there was no clear function for this protein. , 9-stranded anti-parallel β-sheets, each flanked at its N-terminus by one pair of parallel α-helices and at its C-terminus by a set of three α-helices in an helix-loop-helix-loop-helix configuration (Fig. 2a). The structure clearly indicated that ChuS arose by gene duplication, as can be seen by on each other resulting in an RMS of of 2.1 Å (Fig. 2b). Based on the ChuS site directed mutation studies were carried out that indicated that demonstrate in the ChuS-heme complex that His193 is responsible for axial ligation of heme, and that Arg100 further stabilizes the heme group from the medial side of the protein via two water molecules (Fig. 3). It is also interesting to note that the dimer structure of ChuX displays a surprising degree of similarity to the monomer structures of two other heme utilization operon proteins, E. coli ChuS and Y. enterocolitica HemS, both of which have an internal domain repeat (Fig. 4).