Pekkala Mira, Hieta Reija, Bergmann Ulrich, Kivirikko Kari I, Wierenga Rik K, Myllyharju Johanna
Department of Biochemistry and Biocenter Oulu and Collagen Research Unit, University of Oulu, FIN-90014 Oulu, Finland.
J Biol Chem. 2004 Dec 10;279(50):52255-61. doi: 10.1074/jbc.M410007200. Epub 2004 Sep 28.
Collagen prolyl 4-hydroxylases catalyze the formation of 4-hydroxyproline in -X-Pro-Gly-sequences and have an essential role in collagen synthesis. The vertebrate enzymes are alpha2beta2 tetramers in which the catalytic alpha-subunits contain separate peptide-substrate-binding and catalytic domains. We report on the crystal structure of the peptide-substrate-binding domain of the human type I enzyme refined at 2.3 A resolution. It was found to belong to a family of tetratricopeptide repeat domains that are involved in many protein-protein interactions and consist of five alpha-helices forming two tetratricopeptide repeat motifs plus the solvating helix. A prominent feature of its concave surface is a deep groove lined by tyrosines, a putative binding site for proline-rich Tripeptides. Solvent-exposed side chains of three of the tyrosines have a repeat distance similar to that of a poly-L-proline type II helix. The aromatic surface ends at one of the tyrosines, where the groove curves almost 90 degrees away from the linear arrangement of the three tyrosine side chains, possibly inducing a bent conformation in the bound peptide. This finding is consistent with previous suggestions by others that a minimal structural requirement for proline 4-hydroxylation may be a sequence in the poly-L-proline type II conformation followed by a beta-turn in the Pro-Gly segment. Site-directed mutagenesis indicated that none of the tyrosines was critical for tetramer assembly, whereas most of them were critical for the binding of a peptide substrate and inhibitor both to the domain and the alpha2beta2 enzyme tetramer.
胶原蛋白脯氨酰4-羟化酶催化在-X-Pro-Gly-序列中形成4-羟脯氨酸，在胶原蛋白合成中起关键作用。脊椎动物的这种酶是α2β2四聚体，其中催化性的α亚基包含独立的肽底物结合结构域和催化结构域。我们报道了人类I型酶的肽底物结合结构域的晶体结构，其分辨率为2.3埃。发现它属于四肽重复结构域家族，该家族参与许多蛋白质-蛋白质相互作用，由五个α螺旋组成两个四肽重复基序以及溶剂化螺旋。其凹面的一个显著特征是由酪氨酸排列形成的深沟，这是富含脯氨酸的三肽的假定结合位点。三个酪氨酸的溶剂暴露侧链具有与聚-L-脯氨酸II型螺旋相似的重复距离。芳香表面在其中一个酪氨酸处终止，此处沟从三个酪氨酸侧链的线性排列弯曲近90度，可能诱导结合肽中的弯曲构象。这一发现与其他人之前的建议一致，即脯氨酸4-羟化的最小结构要求可能是聚-L-脯氨酸II型构象中的一个序列，随后是Pro-Gly片段中的一个β转角。定点诱变表明，没有一个酪氨酸对四聚体组装至关重要，而大多数酪氨酸对肽底物和抑制剂与该结构域以及α2β2酶四聚体的结合至关重要。
