Conformational specificity in allosteric signaling: high‐throughput measurement of modular secondary structural changes within human Eg5 kinesin

Abstract

The motor domain of human Eg5 kinesin (HsEg5) displays an allosteric pocket at the extracellular surface; binding of monastrol or S‐trityl‐L‐cysteine (STC) to this allosteric site, created by the L5 loop, and subsequent distal conformational changes impairs ATP hydrolysis and cellular function. We examined the roles of E116 and E118 in the L5 loop in allosteric signaling. First, there is a positional correlation on the outcome of amino acid substitutions, irrespective of its nature. 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 kinesin L5 loop can drive upregulation and downregulation of ATP hydrolysis, respectively. From high‐throughput measurements of the secondary structure composition of proteins in solution, vibrational signatures from upregulated proteins were distinct from the net changes measured from downregulated proteins. Moreover, spectral changes found for E116 variants were similar to those of wildtype Eg5 with monastrol or STC. Therefore, allosteric inhibition by amino acid substitution or by small molecules both result in convergent steady‐state changes to the secondary structure of the Eg5 motor domain in solution, and these rapid methods will provide insight into how long‐range structural changes impact motor function.