Affinity- and specificity-enhancing mutations are frequent in multispecific interactions between TIMP2 and MMPs.
Multispecific proteins play a major role in controlling various functions such as signaling, regulation of transcription/translation, and immune response. Hence, a thorough understanding of the atomic-level principles governing multispecific interactions is important not only for the advancement of basic science but also for applied research such as drug design. Here, we study evolution of an exemplary multispecific protein, a Tissue Inhibitor of Matrix Metalloproteinases 2 (TIMP2) that binds with comparable affinities to more than twenty-six members of the Matrix Metalloproteinase (MMP) and the related ADAMs families. We postulate that due to its multispecific nature, TIMP2 is not optimized to bind to any individual MMP type, but rather embodies a compromise required for interactions with all MMPs. To explore this hypothesis, we perform computational saturation mutagenesis of the TIMP2 binding interface and predict changes in free energy of binding to eight MMP targets. Computational results reveal the non-optimality of the TIMP2 binding interface for all studied proteins, identifying many affinity-enhancing mutations at multiple positions. Several TIMP2 point mutants predicted to enhance binding affinity and/or binding specificity towards MMP14 were selected for experimental verification. Experimental results show high abundance of affinity-enhancing mutations in TIMP2, with some point mutations producing more than ten-fold improvement in affinity to MMP14. Our computational and experimental results collaboratively demonstrate that the TIMP2 sequence lies far from the fitness maximum when interacting with its target enzymes. This non-optimality of the binding interface and high potential for improvement might characterize all proteins evolved for binding to multiple targets.
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Oz
O
Sharabi
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Jason
J
Shirian
Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel.
Moran
M
Grossman
Wolfson Center for Structural Biology, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Mario
M
Lebendiker
Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel.
Irit
I
Sagi
Department of Biological Chemistry, The Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem, Israel.
Julia
J
Shifman
D019943:Amino Acid Substitution; D006801:Humans; D053511:Matrix Metalloproteinase 14 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D016296:Mutagenesis; D017354:Point Mutation; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
C560823:TIMP2 protein, human; D019716:Tissue Inhibitor of Metalloproteinase-2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14