Gregory Benison

Determination of Protein Backbone Structures from Residual Dipolar Couplings

There are a number of circumstances in which a focus on determination of the backbone structure of a protein, as opposed to a complete all-atom structure, may be appropriate. This is particularly the case for structures determined as a part of a structural genomics initiative in which computational modeling of many sequentially related structures from the backbone of a single family representative is anticipated. It is, however, also the case when the backbone may be a stepping-stone to more targeted studies of ligand interaction or protein-protein interaction. Here an NMR protocol is described that can produce a backbone structure of a protein without the need for extensive experiments directed at side chain resonance assignment or the collection of structural information on side chains. The procedure relies primarily on orientational constraints from residual dipolar couplings as opposed to distance constraints from NOEs. Procedures for sample preparation, data acquisition, and data analysis are described, along with examples from application to small target proteins of a structural genomics project.

Potential role for phosphorylation in differential regulation of the assembly of dynein light chains

The homodimeric light chains LC8 and Tctex-1 are integral parts of the microtubule motor cytoplasmic dynein, as they directly associate with dynein intermediate chain IC and various cellular cargoes. These light chains appear to regulate assembly of the dynein complex by binding to and promoting dimerization of IC. In addition, both LC8 and Tctex-1 play roles in signaling, apoptosis, and neuronal development that are independent of their function in dynein, but it is unclear how these various activities are modulated. Both light chains undergo specific phosphorylation, and here we present biochemical and NMR analyses of phosphomimetic mutants that indicate how phosphorylation may regulate light chain function. For both LC8 and Tctex-1, phosphorylation promotes dissociation from IC while retaining their binding activity with other non-dynein proteins. Although LC8 and Tctex-1 are homologs having a common fold, their reduced affinity for IC upon phosphorylation arises by different mechanisms. In the case of Tctex-1, phosphorylation directly masks the IC binding site at the dimer interface, whereas for LC8, phosphorylation dissociates the dimer and indirectly eliminates the binding site. This modulation of the monomer-dimer equilibrium by phosphorylation provides a novel mechanism for discrimination among LC8 binding partners.

The Interplay of Ligand Binding and Quaternary Structure in the Diverse Interactions of Dynein Light Chain LC8.

Dynein light chain LC8 is a small, dimeric, and very highly conserved globular protein that is an integral part of the dynein and myosin molecular motors but appears to have a broader role in multiple protein complexes unrelated to molecular motors. LC8 binds to two families of targets: those having a KXTQT sequence fingerprint and those having a GIQVD fingerprint. All known LC8 binding partners containing these fingerprints share a common binding site on LC8 that raises the question of what determines binding specificity. Here, we present the crystal structure of apo-LC8 at 1.7-A resolution, which, when compared with the crystal structures of several LC8 complexes, gives insight into the mechanism underlying the binding diversity of LC8. Peptide binding is associated with a shift in quaternary structure that expands the hydrophobic binding surface available to the ligand, in addition to changes in tertiary structure and ordering of LC8 around the binding groove. The observed quaternary shift suggests a mechanism by which binding at one of the two identical sites can influence binding at the other. NMR spectra of titrations with peptides from each fingerprint family show evidence of allosteric interaction between the two binding sites, to a differing degree in the two ligand families. Allosteric interaction between the binding sites may be a mechanism to promote simultaneous binding of ligands from the same family, providing a physiological role for the two fingerprints.

Structural, thermodynamic, and kinetic effects of a phosphomimetic mutation in dynein light chain LC8.

Dynein light chain LC8 is a small, dimeric, very highly conserved globular protein first identified as an integral part of the dynein and myosin molecular motors but now recognized as a dimerization hub with wider significance. Phosphorylation at Ser88 is thought to be involved in regulating LC8 in the apoptotic pathway. The phosphomimetic Ser88Glu mutation weakens dimerization of LC8 and thus its overall ligand-binding affinity, because only the dimer binds ligands. The 1.9 A resolution crystal structure of dimeric LC8(S88E) bound to a fragment of the ligand Swallow (Swa) presented here shows that the tertiary structure is identical to that of wild-type LC8/Swa, with Glu88 well accommodated sterically at the dimer interface. NMR longitudinal magnetization exchange spectroscopy reveals remarkably slow association kinetics (k(on) approximately 1 s(-1) mM(-1)) in the monomer-dimer equilibrium of both wild-type LC8 and LC8(S88E), possibly due to the strand-swapped architecture of the dimer. The Ser88Glu mutation raises the dimer dissociation constant (K(D)) through a combination of a higher k(off) and lower k(on). Using a minimal model of titration linked to dimerization, we dissect the thermodynamics of dimerization of wild-type LC8 and LC8(S88E) in their various protonation states. When both Glu88 residues are protonated, the LC8(S88E) dimer is nearly as stable as the wild-type dimer, but deprotonation of one Glu88 residue raises K(D) by a factor of 400. We infer that phosphorylation of one subunit of wild-type LC8 raises K(D) by at least as much to prevent dimerization of LC8 at physiological concentrations. Some LC8 binding partners may bind tightly enough to promote dimerization even when one subunit is phosphorylated; thus linkage between phosphorylation and dimerization provides a mechanism for differential regulation of binding of LC8 to its diverse partners.

NMR Analysis of Dynein Light Chain Dimerization and Interactions With Diverse Ligands

NMR is a powerful tool for quantitative measurement of the thermodynamic properties of biological systems. In this review, we discuss the role NMR has played in understanding the various coupled equilibria in dimerization of dynein light chain LC8 and in its interactions with its ligands. LC8, a very highly conserved 89-residue homodimer also known as DYNLL, is an essential component of the dynein and Myosin V molecular motors and is also found in various other complexes. LC8 binds to disordered segments of its partners, promoting them to dimerize and form more ordered structures, often coiled coils. The monomer-dimer equilibrium is controlled by electrostatic interactions at the dimer interface, such as by phosphorylation of residue Ser88, which is a regulatory mechanism for LC8 in vivo. NMR experiments have uncovered several subtle interactions--weak dimerization of a phosphomimetic mutant, and allosteric interaction between the LC8 binding sites--that have been overlooked by other methods. NMR has also provided a residue-specific view of the titration of histidine residues at the LC8 dimer interface, and of a nascent helix in one of the binding partners, the primarily disordered dynein intermediate chain IC74. We give special attention to methods for quantitative interpretation of NMR spectra, an important consideration when using NMR to measure equilibria.