PDBsum entry 2jd5

Transferase

PDB id: 2jd5

Contents

Protein chains

353 a.a. *

Ligands

ARG-GLU-ARG-SER-PRO

SO4 ×2

Metals

_MG ×2

Waters ×133

* Residue conservation analysis

PDB id:

2jd5

Name:

Transferase

Title:

Sky1p bound to npl3p-derived substrate peptide

Structure:

Serine/threonine-protein kinase sky1. Chain: a, b. Fragment: residues 138-306,539-742. Synonym: sky1p, srpk. Engineered: yes. Nucleolar protein 3. Chain: c. Fragment: unp residues 408-414. Synonym: mitochondrial targeting suppressor 1 protein, nuclear

Source:

Saccharomyces cerevisiae. Baker's yeast. Organism_taxid: 4932. Expressed in: escherichia coli. Expression_system_taxid: 469008. Synthetic: yes. Organism_taxid: 4932

Resolution:

2.50Å R-factor: 0.210 R-free: 0.255

Authors:

B.Nolen,R.Lukasiewicz,J.A.Adams,D.Huang,G.Ghosh

Key ref:

R.Lukasiewicz et al. (2007). The RGG domain of Npl3p recruits Sky1p through docking interactions. J Mol Biol, 367, 249-261. PubMed id: 17239901 DOI: 10.1016/j.jmb.2006.12.031

Date:

04-Jan-07 Release date: 06-Feb-07

Protein chains

Q03656  (SKY1_YEAST) -  Serine/threonine-protein kinase SKY1 from Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

Seq: 742 a.a.

Struc: 353 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.7.11.1 - non-specific serine/threonine protein kinase.
• Reaction:
  1. L-seryl-[protein] + ATP = O-phospho-L-seryl-[protein] + ADP + H⁺
  2. L-threonyl-[protein] + ATP = O-phospho-L-threonyl-[protein] + ADP + H⁺

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2006.12.031

J Mol Biol 367:249-261 (2007)

PubMed id: 17239901

The RGG domain of Npl3p recruits Sky1p through docking interactions.

R.Lukasiewicz, B.Nolen, J.A.Adams, G.Ghosh.

ABSTRACT

The SR protein kinase in yeast, Sky1p, phosphorylates yeast SR-like protein, Npl3p, at a single serine residue located at its C terminus. We report here the X-ray crystal structure of Sky1p bound to a substrate peptide and ADP. Surprisingly, an Npl3p-derived substrate peptide occupies a groove 20 A away from the kinase active site. In vitro studies support the substrate-docking role of this groove. Mutagenesis and binding studies reveal that multiple degenerate short peptide motifs located within the RGG domain of Npl3p serve as the substrate docking motifs. However, a single docking motif is sufficient for its stable interaction with the kinase. Methylation of the docking motifs abolishes kinase binding and phosphorylation of Npl3p. Remarkably, removal of the docking groove in the kinase or the docking motifs of the substrate does not reduce the overall catalytic efficiency of the phosphorylation reaction in any significant manner. We suggest that docking interaction between Sky1p and Npl3p is essential for substrate recruitment and binding specificity.

Selected figure(s)

Figure 1.
Figure 1. An Npl3p-derived peptide binds Sky1p in a region distal to the active site. (a) Domain maps of Sky1p and Npl3p. Full-length Sky1p contains two kinase sub-domains (blue) bifurcated by a spacer region (red). The Sky1p kinase domain is also flanked by non-kinase core regions (white). A minimal construct of Sky1p, Sky1pΔN(137)ΔS was used for crystallization. Yellow rectangle denotes poly-histidine tag. The RGG domain of Npl3p (red) contains eight RS dipeptide repeats, the last of which is phosphorylated by Sky1p (denoted by yellow-green). The serine at position 4 of the peptide corresponds to the phosphorylated serine. (b) Surface rendition of Sky1p/peptide/ADP complex. The peptide and ADP are shown in yellow and SKy1p in blue. (c) 2F[o]−F[c] electron density map (1σ) for both the Npl3p-derived peptide (left molecule) and ADP (right molecule) binding. (d) An overlay of the small lobes (residues 144−300) of the apo-kinase (blue) and the peptide-bound kinase (red) shows that the kinase structure remains mostly unchanged as a substrate peptide binds distal to the active site.

Figure 2.
Figure 2. Sky1p and SRPK1 share a conserved substrate docking groove. (a) Residues in Sky1p docking groove involved in the interaction with the peptide/ADP composite substrate. Residues labeled in cyan (I653, K657and W659) and red (K668) were mutated to alanine in the Sky1p QM mutant. Green (D601, D617 and E624) and red residues (K668) were mutated to alanine in the Sky1p 4M mutant. Residues labeled in blue (L603, Y612, Q621 and V664) make non-specific interactions with either the peptide or ADP. Note that residues D617 and E624 form hydrogen bonds with the adenine ring of ADP. (b) Interactions between SRPK1 docking groove and a similar peptide as in (a) are shown. (c) The sequence alignment of Sky1p and SRPK1 docking grooves shows that the region is highly conserved between Sky1p and SRPK1. Identical residues are shaded. (d) Kinetic analysis shows that the docking groove of Sky1p is essential for substrate phosphorylation.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2007, 367, 249-261) copyright 2007.

Figures were selected by an automated process.