Heteronuclear NMR Identifies a Nascent Helix in Intrinsically Disordered Dynein Intermediate Chain: Implications for Folding and Dimerization - Analytical Methods & Tools for Lyophilization

Heteronuclear NMR Identifies a Nascent Helix in Intrinsically Disordered Dynein Intermediate Chain: Implications for Folding and Dimerization
Received 16 June 2006; revised 29 July 2006; accepted 1 August 2006. Edited by M. Moody. Available online 4 August 2006.
Gregory Benison?, a, Afua Nyarko?, 1, a and Elisar Barbar, a
Journal of Molecular Biology
Volume 362, Issue 5 , 6 October 2006
ScienceDirect
Copyright ? 2006 Elsevier Ltd All rights reserved.
aDepartment of Biochemistry and Biophysics, Oregon State University, Corvallis, OR 97331, USA
Abstract
The intermediate chain of dynein forms a tight subcomplex with dimeric light chains LC8 and Tctex-1, and together they constitute the cargo attachment complex. There is considerable interest in identifying the role of these light chains in the assembly of the two copies of the intermediate chain. The N-terminal domain of the intermediate chain, IC1-289, contains the binding sites for the light chains, and is a highly disordered monomer but gains helical structure upon binding to light chains LC8 and Tctex-1. To provide insights into the structural and dynamic changes that occur in the intermediate chain upon light chains binding, we have used NMR spectroscopy to compare the properties of two distinct sub-domains of IC1-289: IC84-143 which is the light chains binding domain, and IC198-237, which contains a predicted coiled coil necessary for the increase in ordered structure upon light chain binding. Neither construct has stable secondary structure when probed by circular dichroism and amide chemical shift dispersion. Specific residues of IC84-143 involved in binding to the light chains were identified by their increase in resonance line broadening and the corresponding large intensity reduction in 1H-15N HSQC spectra. Interestingly, IC84-143 shows no sign of structure formation after binding to either LC8 or Tctex-1 or to both. IC198-237, on the other hand, contains a population of a nascent helix at low temperature as identified by heteronuclear NMR relaxation measurements, secondary chemical shifts, and sequential amide-amide connectivities. These data are consistent with a model for light chain binding coupled to intermediate chain dimerization through forming a coiled coil distant from the binding site.
Keywords: dynein intermediate chain; dynein light chain; folding coupled to binding; intrinsically unfolded proteins; nascent helix
Abbreviations: IC74, 74 kDa intermediate chain subunit of cytoplasmic dynein; IC1-289, IC114-260, IC84-143, IC198-237, constructs of the intermediate chain subunit which include residues 1 to 289, 114 to 260, 84 to 143 and 198 to 237, respectively; LC8, 10 kDa dynein light chain; Tctex-1, 12.5 kDa dynein light chain; CD, circular dichroism; HSQC, heteronuclear single quantum correlation spectroscopy; DSS, 2,2-dimethyl-2-silapentane-5-sulfonic acid; NOE, nuclear Overhauser effect; CSI, chemical shift index

Corresponding author.
? A.N. and G.B. contributed equally to this work.
1 Present address: A. Nyarko, Department of Biology, University of York, YO10 5YW, UK.
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