The crystal structure determination of E. coli CobW was undertaken as part of a structural genomics effort (http://s2f.carb.nist.gov
) in order to assist with the functional assignment of the protein. The CobW protein from E. coli was cloned, expressed, and the crystal structure determined at 2.4- resolution. The CobW protein is folded into two distinct domains with a 22-residue linker between them. The N-terminal domain includes residues 1-202 and is dominated by a 9-stranded mostly parallel b-sheet. The fold is characteristic of P-loop NTP-binding proteins (Saraste et al., 1990). The P-loop with the consensus sequence G11XXXXGKT18 follows the N-terminal b-strand, which is located in the middle of the b-sheet. The distribution of conserved residues reveals a cluster at the NTP-binding site that includes the four canonical motifs of G-proteins, namely: (i) the phosphate-binding Walker A motif (P-loop), (ii) the Walker B motif with the invariant residue Glu97, (iii) the switch I region with the invariant residues Asn38 and Glu39 that bind the g-phosphate of NTP, and (iv) the base recognition motif T158KTD161 (usually NKXD in the G-proteins) with the invariant residues Lys159 and Asp161. The presence of the latter motif indicates the specificity of CobW towards guanine. Specific binding of GTP by CobW was established in a series of experiments using a nucleotide analogue with the fluorescent label trinitrophenyl (TNP) attached to the ribose of GTP. While the crystal structure suggests the molecular function of CobW as a GTP-dependent regulator, the biological role of the protein in the cell remains unclear. Analysis of the gene expression levels in E. coli under the conditions of genotoxic stress indicates that CobW may regulate the pathway that is essential for cell viability. The cobW gene was identified as part of the cob cluster of genes in Pseudomonas denitrificans and therefore may be somehow involved in cobalamin biosynthesis. Given the conserved motif GCXCC in the b-meander, the metallochaperone function is a distinct possibility. It is worth to note that two other GTPases closely related to CobW, UreG and HypB, have been implicated in the assembly of the Ni2+ centers in metalloenzymes. Further biochemical and biophysical studies will shed more light on the molecular and cellular functions of the CobW protein. These studies will be facilitated by the three-dimensional structure of CobW.