Conformational Specificity in Allosteric Signaling: High-throughput Measurement of Modular Secondary Structural Changes within Human Eg5 Kinesin

Abstract

Communication within allosteric enzymes requires long-range interactions. The ATPase activity of human Eg5 kinesin (HsEg5) is allosterically inhibited upon noncovalent binding of monastrol or S-trityl-L-cysteine (STC) with the L5 loop and subsequent distal conformation changes. Our hypothesis is that the E116 and E118 carboxylates, flanking an isolated β-bridge in the L5 loop, are required for allosteric signaling during small chemical inhibition. Surprisingly, assessment by molecular and biochemical methods revealed that substitution of these carboxylates, irrespective of its nature, has a positional dependence on HsEg5 ATPase rates. Single-site substitution of E118 increased basal ATP hydrolysis rates of Eg5, whereas substitution of E116 resulted in lower ATPase rates. Thus, sequence variation at residues 116 and 118 of the L5 loop can drive upregulation and downregulation of ATP hydrolysis in vitro, respectively. We conducted high-throughput infrared measurements of the secondary structure composition of the upregulated and downregulated proteins in solution. Vibrational signatures from upregulated proteins were distinct from the net changes measured from downregulated proteins. Compared to control samples, in HsEg5 motors with E118 substitutions, amide I′ components between 1642-1620 cm−1 decreased in normalized area, whereas proteins with alterations of E116 exhibited an increased contribution in the amide I′ area for modes between 1684 and 1646 cm−1. Moreover, the spectral changes exhibited by E116 variants were similar to those of wildtype HsEg5 during allosteric inhibition by monastrol or STC. We conclude that allosteric inhibition by amino acid substitution or by small molecules both result in convergent steady-state changes to the secondary structure of the HsEg5 motor domain in solution, and these rapid methods will provide insight into how long-range structural changes impact motor function.