PDBsum entry: 2p1r

Transferase

PDB id: 2p1r

Contents

Protein chains

290 a.a. *

Metals

_CA ×10

_CL ×2

_NA ×2

Waters ×414

* Residue conservation analysis

PDB id:

2p1r

Name:

Transferase

Title:

Crystal structure of salmonella typhimurium yegs, a putative kinase homologous to eukaryotic sphingosine and diacylglyce kinases.

Structure:

Lipid kinase yegs. Chain: a, b, c, d. Engineered: yes

Source:

Salmonella typhimurium. Organism_taxid: 99287. Strain: lt2. Gene: yegs. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

2.50Å R-factor: 0.206 R-free: 0.255

Authors:

C.E.Nichols,H.K.Lamb,M.Lockyer,I.G.Charles,S.Pyne,A.R.Hawkin D.K.Stammers

Key ref:

C.E.Nichols et al. (2007). Characterization of Salmonella typhimurium YegS, a putative lipid kinase homologous to eukaryotic sphingosine and diacylglycerol kinases. Proteins, 68, 13-25. PubMed id: 17393457 DOI: 10.1002/prot.21386

Date:

06-Mar-07 Release date: 23-Oct-07

Protein chains

Q8ZNP1  (YEGS_SALTY) -  Probable lipid kinase YegS

Seq: 299 a.a.

Struc: 290 a.a.

 Gene Ontology (GO) functional annotation 

• Cellular component: cytoplasm (1 term)
• Biological process: phospholipid biosynthetic process (4 terms)
• Biochemical function: nucleotide binding (8 terms)

DOI no: 10.1002/prot.21386

Proteins 68:13-25 (2007)

PubMed id: 17393457

Characterization of Salmonella typhimurium YegS, a putative lipid kinase homologous to eukaryotic sphingosine and diacylglycerol kinases.

C.E.Nichols, H.K.Lamb, M.Lockyer, I.G.Charles, S.Pyne, A.R.Hawkins, D.K.Stammers.

ABSTRACT

Salmonella typhimurium YegS is a protein conserved in many prokaryotes. Although the function of YegS is not definitively known, it has been annotated as a potential diacylglycerol or sphingosine kinase based on sequence similarity with eukaryotic enzymes of known function. To further characterize YegS, we report its purification, biochemical analysis, crystallization, and structure determination. The crystal structure of YegS reveals a two-domain fold related to bacterial polyphosphate/ATP NAD kinases, comprising a central cleft between an N-terminal alpha/beta domain and a C-terminal two-layer beta-sandwich domain; conserved structural features are consistent with nucleotide binding within the cleft. The N-terminal and C-terminal domains of YegS are however counter-rotated, relative to the polyphosphate/ATP NAD kinase archetype, such that the potential nucleotide binding site is blocked. There are also two Ca2+ binding sites and two hydrophobic clefts, one in each domain of YegS. Analysis of mutagenesis data from eukaryotic homologues of YegS suggest that the N-terminal cleft may bind activating lipids while the C-terminal cleft may bind the lipid substrate. Microcalorimetry experiments showed interaction between recombinant YegS and Mg2+, Ca2+, and Mn2+ ions, with a weaker interaction also observed with polyphosphates and ATP. However, biochemical assays showed that recombinant YegS is endogenously neither an active diacylglycerol nor sphingosine kinase. Thus although the bioinformatics analysis and structure of YegS indicate that many of the ligand recognition determinants for lipid kinase activity are present, the absence of such activity may be due to specificity for a different lipid substrate or the requirement for activation by an, as yet, undetermined mechanism. In this regard the specific interaction of YegS with the periplasmic chaperone OmpH, which we demonstrate from pulldown experiments, may be of significance. Such an interaction suggests that YegS can be translocated to the periplasm and directed to the outer-membrane, an environment that may be required for enzyme activity.

Selected figure(s)

Figure 3.
Figure 3. (a, b) VMD generated New-Cartoon format illustrations of YegS colored as for Figure 2(d): (a) Close-up view of Ca-site 1 and (b) close-up view of Ca-site 2 with contacting residues marked in each case. (c) multiple overlay of available polyphosphate/ATP NAD kinase structures (yellow, red, and purple) with YegS (green). (d) Cartoon format overlay of YegS (green) and PDB 1Z0S (A. fulgidus polyphosphate/ATP NAD kinase, purple); Mg^2+ and ATP/pyrophosphate ligands from 1Z0S are also shown (VDW and licorice format respectively). This alignment was performed using the N-terminal domains only and clearly illustrates the overlap of the YegS C-terminal domain with the ATP binding site of 1Z0S.

Figure 4.
Figure 4. (a) C-terminal domain alignment of YegS chain-A (green) and PDB 1Z0U (A. fulgidus polyphosphate/ATP NAD kinase, NADP bound), illustrating the overlap of the YegS insertion domains L2/L4 and the adjacent monomer of 1Z0U which forms the other half of the NAD binding site in this protein. (b) HINGEFIND calculation of effective arc of rotation implied by two-domain superposition of YegS and PDB 1Z0S. The C-terminal YegS domain was fixed and is colored blue, the YegS N-terminal domain crystallographic coordinates are shown in green and the rotated YegS N-terminal domain in tan. YegS Ca-sites 1-2 and the potential ATP-binding cleft predicted by comparison with 1Z0S are also marked. (c, d) VMD generated SURF plot with 1.1 Å probe radius: (c) close-up view of the YegS N-terminal domain showing the conserved hydrophobic cleft, which is suggested as a potential binding cleft for activating lipids and (d) close up view of the YegS C-terminal domain showing the hydrophobic channel running from the predicted ATP binding site (pyrophosphate binding motifs PP1-PP2) all the way to Ca-site 2. This channel overlaps the NAD binding site in 1Z0U and is suggested as the most probable binding site for lipid substrate.

The above figures are reprinted by permission from John Wiley & Sons, Inc.: Proteins (2007, 68, 13-25) copyright 2007.

Figures were selected by the author.