PDBsum entry 3dvh

Motor protein

PDB id: 3dvh

Contents

Protein chains

85 a.a. *

Waters ×106

* Residue conservation analysis

PDB id:

3dvh

Name:

Motor protein

Title:

Lc8 point mutant k36p

Structure:

Dynein light chain 1, cytoplasmic. Chain: a, b, c. Synonym: 8 kda dynein light chain, cut up protein. Engineered: yes. Mutation: yes

Source:

Drosophila melanogaster. Fruit fly. Organism_taxid: 7227. Gene: ctp, cdlc1, ddlc1, cg6998. Expressed in: escherichia coli.

Resolution:

2.00Å R-factor: 0.204 R-free: 0.244

Authors:

C.M.Lightcap,J.C.Williams

Key ref:

C.M.Lightcap et al. (2008). Biochemical and structural characterization of the Pak1-LC8 interaction. J Biol Chem, 283, 27314-27324. PubMed id: 18650427 DOI: 10.1074/jbc.M800758200

Date:

18-Jul-08 Release date: 20-Jan-09

Protein chains

Q24117  (DYL1_DROME) -  Dynein light chain 1, cytoplasmic from Drosophila melanogaster

Seq: 89 a.a.

Struc: 85 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

DOI no: 10.1074/jbc.M800758200

J Biol Chem 283:27314-27324 (2008)

PubMed id: 18650427

Biochemical and structural characterization of the Pak1-LC8 interaction.

C.M.Lightcap, S.Sun, J.D.Lear, U.Rodeck, T.Polenova, J.C.Williams.

ABSTRACT

Pak1 (p21-activated kinase-1) and the dynein light chain, LC8, are overexpressed in breast cancer, and their direct interaction has been proposed to regulate tumor cell survival. These effects have been attributed in part to Pak1-mediated phosphorylation of LC8 at serine 88. However, LC8 is homodimeric, which renders Ser(88) inaccessible. Moreover, Pak1 does not contain a canonical LC8 binding sequence compared with other characterized LC8 binding sequences. Together, these observations raise the question whether the Pak1/LC8 interaction is distinct (i.e. enabled by a unique interface independent of LC8 dimerization). Herein, we present results from biochemical, NMR, and crystallographic studies that show that Pak1 (residues 212-222) binds to LC8 along the same groove as canonical LC8 interaction partners (e.g. nNOS and BimL). Using LC8 point mutants K36P and T67A, we were able to differentiate Pak1 from canonical LC8 binding sequences and identify a key hydrogen bond network that compensates for the loss of the conserved glutamine in the consensus sequence. We also show that the target binding interface formed through LC8 dimerization is required to bind to Pak1 and precludes phosphorylation of LC8 at Ser(88). Consistent with this observation, in vitro phosphorylation assays using activated Pak1 fail to phosphorylate LC8. Although these results define structural details of the Pak1/LC8 interaction and suggest a hierarchy of target binding affinities, they do not support the current model whereby Pak1 binds to and subsequently phosphorylates LC8 to promote anchorage-independent growth. Rather, they suggest that LC8 binding modulates Pak1 activity and/or nuclear localization.

Selected figure(s)

Figure 3.
Mutational analysis of Pak1 binding sequence. Analytical SEC of WT-LC8 (A) and individual point mutants (B-K) in SMT3-Pak1-(204-226) was used to qualitatively assess the role of each residue in the LC8-binding region of Pak1. SDS-PAGE of fractions eluting between 10.1 and 10.5 ml is shown for each experiment. The expected position of LC8 through SDS-PAGE is indicated with an asterisk. Each experiment was performed three times.

Figure 7.
Role of Thr^67 on LC8-Pak1 interaction. A, stereoview of the LC8-Pak1 hydrogen bond network. A conserved hydrogen bond network in LC8 is formed through the side chains of Lys^43, Asp^47, and Thr^67′ and the backbone of Trp^54 in LC8 and interacts with Pak1 through the side chain of Asp^216 through a short hydrogen bond (dotted red lines). B and C, analysis of T67A mutant by analytical SEC. Analytical SEC assays demonstrate that mutation of LC8-Thr^67 to alanine abrogates the Pak1-LC8 interaction. The dynein intermediate chain, on the other hand, encodes threonine at the same position (Asp^216 in Pak1) and does not form a significant hydrogen bond (distance is 5.54 Å based on Protein Data Bank entry 2PG1). Analytical SEC assays show that the dynein IC binds to the mutant, T67A-LC8.

The above figures are reprinted from an Open Access publication published by the ASBMB: J Biol Chem (2008, 283, 27314-27324) copyright 2008.

Figures were selected by an automated process.