The Crystal Structure of Dynein Intermediate Chain-Light Chain Roadblock Complex Gives New Insights into Dynein Assembly

Elisar Barbar,Justin Hall,Yujuan Song,P. Andrew Karplus

doi:10.1074/jbc.m110.103861

Abstract

The roadblock/LC7 dynein light chain is a ubiquitous component of all dyneins and is essential for many diverse processes including proper axonal transport and dendrite growth. In addition, LC7 functions in non-dynein transcriptional activation of the transforming growth factor-beta complex. Crystal structures of Drosophila melanogaster LC7 in the apo form and in complex with a segment of the disordered N-terminal domain of dynein intermediate chain (IC) provide the first definitive identification of the IC sequence recognized by LC7. The site, confirmed by isothermal titration calorimetry studies, overlaps the IC sequence considered in the literature to be an IC self-association domain. The IC peptide binds as two amphipathic helices that lie along an extensive hydrophobic cleft on LC7 and ends with a polar side-chain interaction network that includes conserved residues from both proteins. The LC7 recognition sequence on IC and its interface with LC7 are well conserved and are, thus, likely representative of all IC x LC7 structures. Interestingly, the position of bound IC in the IC x LC7 complex mimics a helix that is integrated into the primary structure in distantly related LC7 homologs. The IC x LC7 structure further shows that the naturally occurring robl(Z) deletion mutation contains the majority of the IC binding site and suggests that promotion of IC binding by phosphorylation of LC7 is an indirect effect.

Highlights

Dynein heavy chains are responsible for motor activity, whereas intermediate chain (IC)2 and light chain subunits comprise the cargo attachment complex
Dlc90F; LC8, the 10-kDa dynein light chain corresponding to gene Cdlc2; LC7, the 11-kDa dynein light chain corresponding to gene robl; roblZ, truncation mutant of D. melanogaster LC7; PDB, Protein Data Bank; Mgl, homodimeric gliding protein MglB from Thermus thermophilus; MP11⁄7p14, mitogen-activated protein kinase interacting heterodimer; ITC, isothermal titration calorimetry
As part of our ongoing effort to elucidate structure-function relationships of dynein light chains and build a comprehensive understanding of dynein assembly, we report here the first molecular-level structure of an IC1⁄7LC7 complex from D. melanogaster (79% sequence identity to H. sapiens LC7)

Summary

Crystal Structure of a Dynein Intermediate Chain Complex

Share a common structural fold of five ␤-strands and three ␣-helices, except the C-terminal ␣-helix is missing in LC7. LC7 apparently has multiple non-dynein interaction partners including the Rab family of GTPase regulators [26], the human reduced folate carrier [27], and the transforming growth factor-␤ receptor complex [28, 29], but in no case is the molecularlevel interaction with a binding partner characterized. Phosphorylation is one mechanism that appears to regulate the multiple roles of LC7. LC7 is serine-phosphorylated after transforming growth factor-␤ receptor activation and binds IC in response to this activation. At present there is no clear molecular-level picture of processes associated with LC7 phosphorylation/dephosphorylation or of its involvement in IC binding. The IC1⁄7LC7 complex structure reveals unexpected insights into dynein assembly and regulation. Comparing LC7 to its likely ancestral fold suggests that the multifunctionality of LC7 is the result of its recruitment for new dynein-related function even while ancient functions are maintained

EXPERIMENTAL PROCEDURES

Recombinant proteins were expressed in Escherichia coli

RMSD from ideal

Syringe Kd

RESULTS

DISCUSSION