The Interface between Catalytic and Hemopexin Domains in Matrix Metalloproteinase-1 Conceals a Collagen Binding Exosite
The Interface between Catalytic and Hemopexin Domains in Matrix Metalloproteinase-1 Conceals a Collagen Binding Exosite
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DOI：
10.1074/jbc.m111.285213
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发表时间：
2011-12-30
影响因子：
4.8
通讯作者：
Pickford, Andrew R.
中科院分区：
文献类型：
--
作者：
Arnold, Laurence H.;Butt, Louise E.;Pickford, Andrew R.
基质金属蛋白酶-1（MMP-1）是胶原蛋白溶解（三螺旋胶原蛋白的分解代谢）的煽动者。先前的研究已经暗示其血红素结合蛋白（HPX）结构域在结合中并且可能使胶原底物不稳定，以准备通过催化（CAT）结构域水解多肽骨架。在这里，我们使用生物物理方法来研究MMP-1 HPX结构域和合成的三螺旋肽（THP）之间形成的复合物，该复合物包含胶原α 1（I）链的MMP-1切割位点。这两种组分通过四叶片HPX结构域推进器的叶片1和2内的结合位点以1：1的化学计量和微摩尔亲和力相互作用。随后的定点诱变和测定表明刀片1残基Phe（301）、瓦尔（319）和Asp（338）参与胶原结合。有趣的是，在全长MMP-1的晶体结构中，Phe（301）被CAT结构域部分掩蔽，这意味着结构域的瞬时分离在胶原识别中是重要的。然而，完整酶中该残基的突变破坏了CAT-HPX界面，导致结合活性急剧降低。因此，这些紧凑和错位状态之间的平衡可能是MMP-1胶原酶活性的基本特征。
Matrix metalloproteinase-1 (MMP-1) is an instigator of collagenolysis, the catabolism of triple helical collagen. Previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilizing the collagen substrate in preparation for hydrolysis of the polypeptide backbone by the catalytic (CAT) domain. Here, we use biophysical methods to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses theMMP-1 cleavage site of the collagen alpha 1(I) chain. The two components interact with 1: 1 stoichiometry and micromolar affinity via a binding site within blades 1 and 2 of the four-bladed HPX domain propeller. Subsequent site-directed mutagenesis and assay implicates blade 1 residues Phe(301), Val(319), and Asp(338) in collagen binding. Intriguingly, Phe(301) is partially masked by the CAT domain in the crystal structure of full-length MMP-1 implying that transient separation of the domains is important in collagen recognition. However, mutation of this residue in the intact enzyme disrupts the CAT-HPX interface resulting in a drastic decrease in binding activity. Thus, a balanced equilibrium between these compact and dislocated states may be an essential feature of MMP-1 collagenase activity.