Insulin dissociates by diverse mechanisms of coupled unfolding and unbinding

Abstract

The protein hormone insulin exists in various oligomeric forms, and a key step in binding its cellular receptor is dissociation of the dimer. This dissociation process and its corresponding association process have come to serve as a paradigms of coupled (un)folding and (un)binding more generally. Despite its fundamental and practical importance, the mechanism of insulin dimer dissociation remains poorly understood.Here, we use molecular dynamics simulations, leveraging recent developments in umbrella sampling, to characterize the energetic and structural features of dissociation in unprecedented detail. We find that the dissociation is inherently multipathway with limiting behaviors corresponding to conformational selection and induced fit, the two prototypical mechanisms of coupled folding and binding. Along one limiting path, the dissociation leads to detachment of the C-terminal segment of the insulin B chain from the protein core, a feature believed to be essential for receptor binding. We simulate IR spectroscopy experiments to aid in interpreting current experiments and identify sites where isotopic labeling can be most effective for distinguishing the contributions of the limiting mechanisms.

Computational IR Spectroscopy of Insulin Dimer Structure and Conformational Heterogeneity

“…Recent simulations of insulin dimer dissociation free energies described a broad distribution of possible energetically favorable dissociation pathways, bounded by two limiting cases: (1) a sequential process of disrupting B-chain α-helix contacts prior to B-chain β contacts (the α path), or (2) β contacts prior to α contacts (β path). 54 To investigate connections between MSM intermediate states and on-path structures during the course of dissociation in that study, we projected the dimer MSM onto collective variables (CVs) describing the dissociation (see Figure S3). As one might expect, the well-solvated β strands of states 45 and 80 lie along the β path when observing CVs involving β contacts, but the α pseudo-dihedral rotation lies closer to the α path in a high free energy region rarely visited in the sampling of dimer dissociation.…”

“…These exhibit twisted motion of the two monomers at the dimer interface, which leads to a disruption of native β-sheet contacts and a reconfiguration of sidechains at the dimer interface. Structural changes along tIC1 are described by several CVs used in previous simulation studies, [53][54] which are summarized in Table 1. The dominant coarse-grained state, the native dimer (N), has the largest population of 66%, an average RMSD value of 4.4 Å, and tIC1 values from 1.0 to 0.77.…”

“…Most prominently the twisting motion is seen through the change in the relative orientation of the two B-chain helices, which can be quantified through an α-helix pseudo-dihedral angle   . 54 The helices rotate relative to each other by -55° on average from 115° to 59° between N and T states. Fig.…”

“…Insulin dimer dissociation is a necessary step prior to binding to insulin receptor, which is regarded as a coupled unfolding and unbinding process studied both computationally and experimentally. [51][52][53][54][55] Insulin monomer is a 51-residue peptide composed of two disulfide bonded chains with 21 residues on chain A and 30 residues on chain B. It has three α-helical segments, a β-turn located from B20Gly to B23Gly, and the B-chain C-terminus ranging from B24Phe-B30Thr is known to be disordered with the extent depending on mutations and solution environment.…”

“…51,[63][64][65] This B-chain C-terminus is also involved in the insulin receptor binding, exhibiting detachment of the B-chain C-terminus, a significant dihedral rotation on B24Phe, and hinging motion upon recognition with the insulin receptor, [66][67][68][69] which may share similar conformational transitions as in the dimerization. 54 Insulin dimer has also become a useful model system for investigating coupled folding and binding dynamics. Computational insulin dimerization studies have focused almost entirely on the conformational characterization in the monomeric state, with the current viewpoint that the dimer structure resembles published crystal structures.…”