Sielecki A R, Fujinaga M, Read R J, James M N
Department of Biochemistry, University of Alberta, Edmonton, Canada.
J Mol Biol. 1991 Jun 20;219(4):671-92. doi: 10.1016/0022-2836(91)90664-r.
The molecular structure of porcine pepsinogen at 1.8 A resolution has been determined by a combination of molecular replacement and multiple isomorphous phasing techniques. The resulting structure was refined by restrained-parameter least-squares methods. The final R factor [formula: see text] is 0.164 for 32,264 reflections with I greater than or equal to sigma (I) in the resolution range of 8.0 to 1.8 A. The model consists of 2785 protein atoms in 370 residues, a phosphoryl group on Ser68 and 238 ordered water molecules. The resulting molecular stereochemistry is consistent with a well-refined crystal structure with co-ordinate accuracy in the range of 0.10 to 0.15 A for the well-ordered regions of the molecule (B less than 15 A2). For the enzyme portion of the zymogen, the root-mean-square difference in C alpha atom co-ordinates with the refined porcine pepsin structure is 0.90 A (284 common atoms) and with the C alpha atoms of penicillopepsin it is 1.63 A (275 common atoms). The additional 44 N-terminal amino acids of the prosegment (Leu1p to Leu44p, using the letter p after the residue number to distinguish the residues of the prosegment) adopt a relatively compact structure consisting of a long beta-strand followed by two approximately orthogonal alpha-helices and a short 3(10)-helix. Intimate contacts, both electrostatic and hydrophobic interactions, are made with residues in the pepsin active site. The N-terminal beta-strand, Leu1p to Leu6p, forms part of the six-stranded beta-sheet common to the aspartic proteinases. In the zymogen the first 13 residues of pepsin, Ile1 to Glu13, adopt a completely different conformation from that of the mature enzyme. The C alpha atom of Ile1 must move approximately 44 A in going from its position in the inactive zymogen to its observed position in active pepsin. Electrostatic interactions of Lys36pN and hydrogen-bonding interactions of Tyr37pOH, and Tyr90H with the two catalytic aspartate groups, Asp32 and Asp215, prevent substrate access to the active site of the zymogen. We have made a detailed comparison of the mammalian pepsinogen fold with the fungal aspartic proteinase fold of penicillopepsin, used for the molecular replacement solution. A structurally derived alignment of the two sequences is presented.
通过分子置换和多同晶型相位技术相结合的方法，已确定了分辨率为1.8埃的猪胃蛋白酶原的分子结构。所得结构通过约束参数最小二乘法进行了优化。对于在8.0至1.8埃分辨率范围内I大于或等于σ（I）的32264个反射，最终的R因子[公式：见正文]为0.164。该模型由370个残基中的2785个蛋白质原子、Ser68上的一个磷酰基和238个有序水分子组成。所得的分子立体化学与一个结构优化良好的晶体结构一致，分子有序区域的坐标精度在0.10至0.15埃范围内（B小于15埃²）。对于酶原的酶部分，其Cα原子坐标与优化后的猪胃蛋白酶结构的均方根差为0.90埃（284个共同原子），与青霉胃蛋白酶的Cα原子坐标的均方根差为1.63埃（275个共同原子）。前肽的额外44个N端氨基酸（Leu1p至Leu44p，在残基编号后使用字母p来区分前肽的残基）采用了一种相对紧凑的结构，由一条长的β链、随后的两个大致正交的α螺旋和一个短的3(10)螺旋组成。与胃蛋白酶活性位点的残基存在紧密的静电和疏水相互作用。N端β链Leu1p至Leu6p构成了天冬氨酸蛋白酶共有的六链β折叠的一部分。在酶原中，胃蛋白酶的前13个残基Ile1至Glu13采用了与成熟酶完全不同的构象。Ile1的Cα原子在从无活性酶原中的位置转变为活性胃蛋白酶中的观察位置时必须移动约44埃。Lys36pN的静电相互作用以及Tyr37pOH和Tyr90H与两个催化天冬氨酸基团Asp32和Asp215的氢键相互作用，阻止了底物进入酶原的活性位点。我们已对哺乳动物胃蛋白酶原折叠与用于分子置换解决方案的青霉胃蛋白酶的真菌天冬氨酸蛋白酶折叠进行了详细比较。给出了两个序列的结构推导比对。
