Structure-function relations of antithrombin III-heparin interactions as assessed by biophysical and biological assays and molecular modeling of peptide-pentasaccharide-docked complexes.
Structure-function relations of antithrombin III-heparin interactions as assessed by biophysical and biological assays and molecular modeling of peptide-pentasaccharide-docked complexes.
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通过生物物理和生物测定以及肽-五糖对接复合物的分子模型评估抗凝血酶 III-肝素相互作用的结构-功能关系。
DOI：
10.1006/abbi.1996.0448
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Harris,RB
中科院分区：
文献类型：
--
作者：
Tyler-Cross,R;Sobel,M;McAdory,LE;Harris,RB
丝氨酸蛋白酶抑制剂抗凝血酶III（ATIII）是内源性凝血的关键调节蛋白。ATIII仅在结合多硫酸化糖胺聚糖（如肝素）后才获得其全部生物活性。肽K121-A134，基于D螺旋区中ATIII的序列，先前由我们显示（Tyler-Crosset al.，Protein Sci.3，620-627，1994）以包含ATIII的部分（或全部）据称的高亲和力肝素结合区。现已制备了一系列肽类似物，其序列与K121-A134相同，只是K121-A134的单个阳离子残基已被Ala连续取代。在一种情况下，对应于ATIII的R129的参考肽的Arg残基已被Gln（R129ΔQ肽）取代，从而模拟天然存在的突变蛋白ATIII日内瓦。通过等温滴定量热法和肽/ATIII竞争结合测定来定量所有肽的肝素亲和力。用Ala替换任何单个阳离子残基对肝素结合具有有害影响。在R129ΔQ肽中观察到肝素亲和力的最大降低（超过30倍）（KD= 1.5 ± 0.06 μMvs参比肽K121-A134的51 ± 2 nM）。此外，每种Ala-替代肽是比参比肽更不有效的ATIII-肝素复合物形成抑制剂。最差的抑制剂是R129ΔQ肽，其显示出抑制效力降低近30%（100 μ M肽的抑制率为60%，参比肽的抑制率为90%）。通过生物测定法测量的肽的相对肝素亲和力与通过滴定量热法测定的相同。因此，我们模拟了五糖单元结构与R129ΔQ肽或参比肽K121-A134之间形成的复合物。在“对接”复合物中，K121-A134的假定构象允许肽的阳离子残基与已知参与结合的五糖的功能性阴离子基团并置。在R129ΔQ肽和五糖之间也可以形成对接复合物，但是观察到关键肽和糖官能团的错配。R129Δ Q-五糖复合物的结构非常不规则，因为F123和Y131完全暴露在表面，可能在水溶液中产生不利的结构。从分子建模的观察使我们能够表明，ATIII日内瓦显示肝素结合亲和力下降，由于其无法形成生产性的结合复合物，其中必要的静电接触之间适当并列的糖阴离子官能团和阳离子氨基酸侧链。
The serine proteinase inhibitor antithrombin III (ATIII) is a key regulatory protein of intrinsic blood coagulation. ATIII attains its full biological activity only upon binding polysulfated glycosaminoglycans, such as heparin. Peptide K121–A134, based on the sequence of ATIII in the D helix region, was previously shown by us (Tyler-Crosset al., Protein Sci.3, 620–627, 1994) to encompass part (or all) of the purported high-affinity heparin binding region of ATIII. A series of peptide analogs has now been prepared whose sequences are identical to K121–A134except that single cationic residues of K121–A134have been successively replaced with Ala. In one case, the Arg residue of the reference peptide corresponding to R129of ATIII has been replaced by Gln (R129ΔQ peptide), thus mimicking the naturally occurring mutant protein, ATIII Geneva. The heparin affinity of all peptides was quantitated by isothermal titration calorimetry and by peptide/ATIII competition binding assays. Replacement of any single cationic residue with Ala had a deleterious effect on heparin binding. The greatest reduction in heparin affinity (more than 30-fold) was observed with the R129ΔQ peptide (KD= 1.5 ± 0.06 μMvs 51 ± 2 nMfor the reference peptide, K121–A134). Furthermore, each of the Ala-replacement peptides was a less-effective inhibitor of ATIII–heparin complex formation than the reference peptide. The poorest inhibitor was the R129ΔQ peptide which showed nearly 30% decrease in inhibition potency (60% inhibition at 100 μMpeptide vs 90% inhibition with the reference peptide). The relative heparin affinities of the peptides measured by biological assay were the same as determined by titration calorimetry. Consequently, we modeled the complexes formed between the pentasaccharide unit structure and the R129ΔQ peptide or the reference peptide, K121–A134. In the “docked” complex, the assumed conformation of K121–A134permitted juxtaposition of the cationic residues of the peptide with functional anionic groups of the pentasaccharide known to be involved in binding. A docked complex could also be formed between the R129ΔQ peptide and the pentasaccharide, but misalignment of critical peptide and saccharide functional groups was observed. The structure of the R129ΔQ–pentasaccharide complex was highly irregular because F123and Y131were completely surface exposed, likely yielding an unfavorable structure in aqueous solution. The observations from molecular modeling allow us to suggest that ATIII Geneva displays decreased heparin binding affinity due to its inability to form a productive binding complex in which essential electrostatic contacts are made between suitably juxtaposed saccharide anionic functional groups and cationic amino acid side chains.