Insights into Affinity Improvement of an Engineered Tissue Inhibitor of Metalloproteinases from Molecular Dynamics Simulations

Abstract

<b>Abstract ID 18265</b> <b>Poster Board 329</b> Matrix MetalloProteinases (MMPs) are a family of 23 multidomain zinc-dependent endopeptidases that primarily target extracellular matrix proteins for degradation. An array of physiological functions rely on MMP activity; dysfunction results in diseases/disorders, including cancers, cardiovascular and pulmonary disease, and arthritis. Despite considerable long-term efforts towards MMP-targeted pharmacology, most potential therapeutics fail in clinical trials due to off-target toxicity against MMPs and other zinc-dependent endopeptidases. Our group has been applying protein engineering efforts towards endogenous Tissue Inhibitors of MetalloProteinases (TIMPs) to create high-affinity and selective inhibitors for disease-associated MMPs. We previously utilized semi-random mutagenesis and yeast surface display to identify an ultrabinder variant of TIMP1 with enhanced affinity towards MMP3, a target in cancer and lung fibrotic diseases, and solved a co-crystal structure of MMP3 bound to this engineered ultrabinder TIMP variant. Here, we employ modeling and molecular dynamics simulations on unbound and MMP3-bound TIMP1 and the ultrabinder variant to investigate the biophysical etiology of MMP-TIMP molecular recognition and of mutation-induced affinity improvements. Our simulations reveal localized induced fit at the zinc-chelating portion of TIMP upon binding MMP, whereas other binding interface regions seem to rely more on innate shape complementarity and conformational selection. Inter-residue motion covariance matrices reveal that MMP binding also reorganizes motion coupling throughout TIMP globally; this suggests that allosteric regulation of the binding interface differs between the unbound and MMP-bound states. Comparisons of molecular dynamics trajectories were made between WT TIMP1 and the ultrabinder TIMP1 variant. Time-resolved analyses of ultrabinder TIMP1 shows that it possesses stable secondary structural alterations that increase TIMP interdomain interactions, as well as protein-protein interactions with MMP3. Dynamical network analyses show that the engineered TIMP1 is able to propagate internal motions with greater efficiency, which appears to suppress motions perturbing TIMP from MMP-congruent configurations. In both the unbound and the MMP-bound states, variant TIMP1 has reduced conformational variability compared to WT TIMP1, with greater representation of conformations resembling that of the bound crystal structure, and more favorable predicted binding energy to MMP3. Overall, our results show that the engineered ultrabinder TIMP1 variant possesses altered protein dynamics that promote MMP3 compatibility in multiple ways, both via preconfiguration of unbound TIMP and via maintenance of the MMP/TIMP complex. These data suggest strategies that utilize intrinsic protein dynamics to improve affinity and selectivity of TIMPs in future engineering efforts towards biopharmaceutical development. This research is supported by NIH R01GM132100