 |
PDBsum entry 2jd5
|
|
|
|
References listed in PDB file
|
 |
|
Key reference
|
|
|
Title
|
|
The rgg domain of npl3p recruits sky1p through docking interactions.
|
|
|
Authors
|
|
R.Lukasiewicz,
B.Nolen,
J.A.Adams,
G.Ghosh.
|
|
|
Ref.
|
|
J Mol Biol, 2007,
367,
249-261.
[DOI no: ]
|
|
|
PubMed id
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
|
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.
|
|
|
|
|
|
|
