Steinmetz M O, Stock A, Schulthess T, Landwehr R, Lustig A, Faix J, Gerisch G, Aebi U, Kammerer R A
M.E.Müller Institute for Microscopy, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.
EMBO J. 1998 Apr 1;17(7):1883-91. doi: 10.1093/emboj/17.7.1883.
We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded approximately 30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I.
我们使用多种重组多肽链片段，研究了盘基网柄菌皮质illin I寡聚化结构域（Ir）组装成紧密堆积的双链平行卷曲螺旋结构的过程。通过圆二色光谱、分析型超速离心和电子显微镜对这些Ir片段的结构进行了分析。缺失作图确定了Ir卷曲螺旋内一个独特的14个残基位点，即Arg311 - Asp324，它是二聚体形成所绝对必需的，这表明仅七肽重复序列不足以形成稳定的卷曲螺旋结构。此外，缺失Ir的六个N端七肽重复序列会导致形成四螺旋而非双螺旋结构，这表明全长皮质illin I卷曲螺旋结构域表现为一个协同折叠单元。最有趣的是，一个包含独特卷曲螺旋“触发”位点Arg311 - Asp324的16个残基肽在水溶液中作为单体时产生了约30%的螺旋形成。pH滴定和NaCl筛选实验表明，该肽的螺旋度强烈依赖于pH和离子强度，这表明肽内带电荷侧链的静电相互作用对于稳定其单体螺旋至关重要。综上所述，这些发现表明Arg311 - Asp324表现为一个自主的螺旋折叠单元，并且这个独特的Ir片段控制着皮质illin I卷曲螺旋形成的过程。
