Anderson Thomas A, Sauer Robert T
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Biophys Chem. 2003;100(1-3):341-50. doi: 10.1016/s0301-4622(02)00291-0.
The Arc repressor of bacteriophage P22 is a member of the ribbon-helix-helix family of transcription factors. Ser32 is a solvent-exposed position that serves a structural role as the N(cap) residue of alpha-helix B of Arc, but also serves a functional role because its side chain is packed close to the sugar-phosphate DNA backbone in the repressor-operator complex. The tolerance of this N(cap) position to amino-acid substitutions was probed by determining the repressor activity in vivo, the thermal stability and the operator-binding activity in vitro of a set of 13 mutant proteins. The stability of position-32 Arc variants, except for Cys32, correlated well with the frequencies observed for the corresponding residues at N(cap) positions in alpha-helices of other proteins. Cysteine was quite stabilizing at the helix-B N(cap) position in Arc, but surprisingly was the least frequent N(cap) residue in the protein database. This latter finding may reflect a hyper-reactivity of N(cap) cysteines, which makes them prone to chemical modification. In general, only Arc variants with small, uncharged residues at position 32 were active in vivo or showed strong operator binding in vitro. Based upon the results presented here, revised sequence alignments of the MetJ and NikR subfamilies with Arc and other ribbon-helix-helix proteins are proposed.
噬菌体P22的Arc阻遏物是转录因子带状-螺旋-螺旋家族的成员。Ser32是一个暴露于溶剂中的位点，它作为Arc的α-螺旋B的N（帽）残基发挥结构作用，但也具有功能作用，因为其侧链在阻遏物-操纵基因复合物中靠近糖-磷酸DNA主链堆积。通过测定一组13种突变蛋白在体内的阻遏活性、热稳定性和体外的操纵基因结合活性，探究了该N（帽）位点对氨基酸取代的耐受性。除Cys32外，32位Arc变体的稳定性与其他蛋白质α-螺旋中N（帽）位点相应残基的观察频率高度相关。半胱氨酸在Arc的螺旋B N（帽）位点相当稳定，但令人惊讶的是，它是蛋白质数据库中最不常见的N（帽）残基。后一发现可能反映了N（帽）半胱氨酸的高反应性，这使得它们易于化学修饰。一般来说，只有在32位带有小的不带电荷残基的Arc变体在体内有活性或在体外表现出强的操纵基因结合能力。基于此处给出的结果，提出了MetJ和NikR亚家族与Arc及其他带状-螺旋-螺旋蛋白的修正序列比对。
