Complex membrane type-1 matrix metalloproteinase (MT1-MMP) with tissue inhibitor of metalloproteinase-1 (TIMP-1)
Publication Abstract from PubMed
Protein flexibility is thought to play key roles during numerous biological processes including antibody affinity maturation, signal transduction and enzyme catalysis. Yet only limited information is available regarding the molecular details linking protein dynamics with function. A single point mutation at the distal site of the endogenous tissue inhibitor of metalloproteinase-1 (TIMP-1) enables this clinical target protein to tightly bind and inhibit membrane type-1 matrix metalloproteinase (MT1-MMP) by increasing only the association constant. The high resolution x-ray structure of this complex determined at 2A could not explain the mechanism of enhanced binding, and pointed to a role for protein conformational dynamics. Molecular dynamics (MD) simulations reveal that the high-affinity TIMP-1 mutants exhibit significantly reduced binding interface flexibility and more stable hydrogen bond networks. This was accompanied by redistribution of the ensemble of substrates to favorable binding conformations that fit the enzyme catalytic site. Apparently, the decrease in backbone flexibility lead to lower entropy cost upon complex formation. This work quantifies the effect of a single point mutation on protein conformational dynamics and function of TIMP-1. Here we argue that controlling intrinsic protein dynamics of MMPs endogenous inhibitors may be utilized for rationalizing the design of selective novel protein inhibitors for this class of enzymes.
Intrinsic protein flexibility of endogenous protease inhibitor TIMP-1 controls its binding interface and effects its function., Grossman M, Tworowski D, Dym O, Lee MH, Levy Y, Murphy G, Sagi I, Biochemistry. 2010 Jun 14. PMID:20545310
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
The human matrix metalloproteinases (MMPs) family comprises a large group of structurally homologous zinc-dependent endopeptidases (e.g. membrane type-1 matrix metalloproteinase (MT1-MMP) (darkmagenta) and membrane type-3 matrix metalloproteinase (MT3-MMP) (magenta), click to see structural comparison) that perform a wide variety of biological roles. In general, the MMPs are inhibited unselectively by all four known tissue inhibitors of metalloproteinases (TIMPs 1-4) which have 40-50% sequence identity. For example, membrane type-3 matrix metalloproteinase (MT3-MMP) can form complex with wild-type TIMP-1 (1uea, colored orange). The WT-TIMP-1 binding interface (cyan) is mainly composed of the N-terminal segment that approaches the active site, the AB loop (Thr33-Tyr35), the CD loop (Ala65-Cys70), and the EF loop (Thr97-Ser100). The pivotal residue, threonine 98 (Thr98), is shown as red sticks. In general, five main chain hydrogen bonds (Cys1-Ser68, Val69-Met66, Gly71-Met66, Cys70-Glu67, and Cys70-Thr98) are intimately involved in the conformational stability of TIMP binding interface when bound to MMP.
Membrane type-1 matrix metalloproteinase (MT1-MMP) (darkmagenta) also forms complex with wild-type TIMP-1 (2j0t, colored orange), producing similar hydrogen bond network in the WT TIMP-1 binding interface as well as in the case with MT3-MMP. This network of hydrogen bonds stabilizes the CD and EF loops that compose the binding interface. Importantly, the hydrogen bond between Cys1 and Ser68 may position the amino and carboxyl groups of Cys1 to effectively coordinate the Zn2+ ion. However, this MT1-MMP-WT-TIMP-1 complex is not tight-binding. MT1-MMP is unique since even though it exhibits high structural homology to all MMPs, it is not inhibited by TIMP-1, but is inhibited by the structural homologous TIMP-2 (1bqq). The single point mutation T98L (mutant TIMP-1 is colored in yellow with T98L shown in red) transformed TIMP-1 into a high affinity inhibitor of MT1-MMP (3ma2). WT-TIMP-1, WT-TIMP-2, and TIMP-1 T98L mutant have kinetic dissociation binding constant (KD) 1.53 x 10-6, 5.61 x 10-8, and 8.70 x 10-8, respectively. So, KD of WT-TIMP-2 is 2 orders of magnitude smaller than that of WT-TIMP-1, indicating the weak affinity between MT1-MMP and WT-TIMP-1. The TIMP-1 T98L mutant regained high-affinity binding to MT1-MMP, resulting in a 2 order of magnitude decrease in KD, similar to the case for WT-TIMP-2, the in vivo inhibitor of MT1-MMP. The overall structures of the complexes of MT1-MMP-WT-TIMP-1 and MT1-MMP-mutant-T98L-TIMP-1 are relatively similar. Even the structure of MT3-MMP-WT-TIMP-1 is similar to those of MT1-MMP-TIMP-1s (with wild-type and TIMP-1 T98L mutant). Leu98 is pointing toward MT1-MMP residues Pro259 and Phe260, establishing a strong hydrophobic core, which is situated near the MT1-MMP catalytic Zn2+ ion surrounded by His239, His243, and His249. So, this T98L replacement may stabilize the entire area by establishing a strong hydrophobic core upon binding to the enzyme. However, it seems unlikely that these additional bonds could
account for the entire binding effect between MT1-MMP and TIMP-1. Statistical analysis of the key hydrogen bond stabilities in the TIMP-1 T98L mutant reveals that the hydrogen bonds network in mutant form is significantly more stable than that in WT-TIMP-1. Mutations that enhance hydrogen
bond stability contribute to the stability of the bound-like, less flexible, conformation of TIMP-1, which eventually results in increasing binding affinity for MT1-MMP. Thus, mutation affected the instrinsic dynamics of the inhibitor rather than its structure, thereby facilitating the interaction.
