A_(21)—Asp人胰岛素突变体2.4埃分辨率晶体结构研究

CRYSTAL STRUCTURE ANALYSIS OF A_(21)Asp-HUMAN INSULIN MUTANT AT 2.4A RESOLUTION

A_(21)-Asp人胰岛素(A_(21)D-HI)是经由蛋白质工程途径制备的突变体,具有高稳定特性.本文报道在2.4埃分辨率A_(21)-HI X-射线晶体结构的测定,所获结构模型经能量制约的最小二乘技术(EREF)精化,最后的晶体学一致性因子R=0.192,共价键长与标准值的平均偏差为0.019埃.在(2F-F_e)电子密度图上,突变残基A_(21)-Asp清晰可见.与天然胰岛素比较,除践基A_(21)外,突变体分子的构象在该分辨率未见显著变化,具有重要结构意义的残基A_(21)-NH与B_(23)-Co之间的主链氢键在两个独立分子中都仍然保持,A链末端羧基与B_(22)-Arg的侧链胍基间的盐键在分子中也仍然保持.在这一基础上,讨论了突变体与分子的化学稳定性和生物活力的关系.

A21 Asp -Human insulin (A21D-HI) is one of the mutants prepared by site - directed mutagenesis,which possesses a high stability (5-7 times as native insulin) in acidic solution. In order to understand the structural basis of such a property and the impact of substitutions at A21, an evolutionally conservative residue, on the molecular structure, crystallographic studies on a series of A21 mutants are undertaken. Here we report the structural analysis of A21D-HI at 2.4A resolution.The crystal structure of A21D -HI was determined by isomorphous difference Fourier method at 2.4A. A cyclic model building on PS390 (Evans & Sutherland) and energy - restrained crystallographic refinement programmed in EREF were involved in the structure analysis. The procedure was terminated with a conventional crystallographic R -value of 0.192 and a r. m. s. boud-length deviation of 0.019A. On the final (2Fo - Fc) map, mutated residue A21 -Asp appeared definitely and showed similar orientations of main-chain and side-chain compared with the native one. Although the positions of both side-chain and terminal carboxyl groups have moved about 1 A. the hydrogen bonds between NH-A,, and CO-B23 are still kept in the two individual molecules. It seems that the chemical stability mainly comes from the substitution of the susceptible group, -NH of asparagine side-chain, and the importance of the residue A21 perhaps mainly involves its main-chain, especially the hydrogen bonded NH - A21 and CO - B23 may have special significance for the performance of insulin activity through a stabilizing role for the β-turn B20- B30 which is crucial for mediating the conformations of B -chain C -terminal segment.The financial support from a UNIDO grant (91/048) is greatly appreciated.