The Structural and Kinetic Ensemble of ASB9's N-Terminus and It's Role in Substrate Recognition

Abstract

Intrinsically disordered proteins (IDPs) have structural ensembles which lack stable tertiary and secondary structure under physiological conditions. IDPs have remolded the structure-function paradigm, often participating in one-to-many and many-to-one protein interactions. One class of IDPs which have remained largely unstudied are the ankyrin repeat and SOCS (supressor of cytokine signaling) box containing proteins (ASBs). This class of proteins have an intrinsically disordered N-terminus, a six ankyrin repeat domain (ARD), and a C-terminal SOCs box domain. The SOCS box of ASB9 is known to interact with elongin B and C and cullin 5 to form E3 ubiquitin ligase complexes, targeting creatine kinases (CK) for ubiquitin mediated degradation. Using isothermal titration calorimetry (ITC) we found the interaction between ASB9 and CK to be largely entropic, with some residues between 19-34 in ASB9 constituting a major portion of ASB9's binding interface. The ARD alone and the N-terminus + ARD of ASB9 have the same entropic contribution to the binding free energy with CK. However, when the first 18 amino acids are removed from ASB9, the entropy of binding to CK is lowered by an order of magnitude. We performed molecular dynamics simulations to rationalize the interaction thermodynamics and explore the structural ensembles of ASB9 (1-252) and CK. These simulations were clustered and dockings were performed on their centroids. Remarkably, residues 23-34 docked into the active site of CK. The docking also found that CK residues 180 to 203 constitute a part of the binding interface, which agree with our hydrogen deuterium exchange mass spectrometry (HDXMS) studies. We plan to precisely characterize the residual dynamics of ASB9 within the complex using NMR spectroscopy interpreted in the light of accelerated molecular dynamics.