Collagens play important roles in development and homeostasis in most higher organisms. In order to function, collagens require the specific chaperone HSP47 for proper folding and secretion. HSP47 is known to bind to the collagen triple helix, but the exact positions and numbers of binding sites are not clear. Here, we employed a collagen II peptide library to characterize high-affinity binding sites for HSP47. We show that many previously predicted binding sites have very low affinities due to the presence of a negatively charged amino acid in the binding motif. In contrast, large hydrophobic amino acids such as phenylalanine at certain positions in the collagen sequence increase binding strength. For further characterization, we determined two crystal structures of HSP47 bound to peptides containing phenylalanine or leucine. These structures deviate significantly from previously published ones in which different collagen sequences were used. They reveal local conformational rearrangements of HSP47 at the binding site to accommodate the large hydrophobic side chain from the middle strand of the collagen triple helix and, most surprisingly, possess an altered binding stoichiometry in the form of a 1:1 complex. This altered stoichiometry is explained by steric collisions with the second HSP47 molecule present in all structures determined thus far caused by the newly introduced large hydrophobic residue placed on the trailing strand. This exemplifies the importance of considering all three sites of homotrimeric collagen as independent interaction surfaces and may provide insight into the formation of higher oligomeric complexes at promiscuous collagen-binding sites.

中文翻译：

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胶原蛋白的一级结构决定了胶原蛋白：HSP47 复合物的化学计量。


胶原蛋白在大多数高等生物的发育和体内平衡中发挥着重要作用。为了发挥作用，胶原蛋白需要特定的伴侣 HSP47 才能正确折叠和分泌。已知 HSP47 与胶原三螺旋结合，但结合位点的确切位置和数量尚不清楚。在这里，我们使用 II 型胶原蛋白肽库来表征 HSP47 的高亲和力结合位点。我们表明，由于结合基序中存在带负电荷的氨基酸，许多先前预测的结合位点具有非常低的亲和力。相反，胶原蛋白序列中某些位置的大疏水性氨基酸（例如苯丙氨酸）会增加结合强度。为了进一步表征，我们确定了与含有苯丙氨酸或亮氨酸的肽结合的 HSP47 的两种晶体结构。这些结构与之前发表的使用不同胶原蛋白序列的结构显着不同。他们揭示了 HSP47 在结合位点的局部构象重排，以适应胶原蛋白三螺旋中链的大疏水侧链，最令人惊讶的是，它们以 1:1 复合物的形式拥有改变的结合化学计量。这种改变的化学计量可以通过与迄今为止确定的所有结构中存在的第二个HSP47分子的空间碰撞来解释，这是由新引入的放置在尾链上的大疏水残基引起的。这例证了将同源三聚胶原蛋白的所有三个位点视为独立相互作用表面的重要性，并且可以深入了解在混杂的胶原蛋白结合位点形成更高的寡聚复合物。