Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains.
Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains.
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DOI：
10.1186/1472-6807-10-13
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发表时间：
2010-05-28
影响因子：
--
通讯作者：
Stammers DK
中科院分区：
文献类型：
--
作者：
Bird LE;Ren J;Nettleship JE;Folkers GE;Owens RJ;Stammers DK
zx1 - 3（锌指和同源盒）形成一组同源基因，编码多结构域蛋白。ZHX蛋白由两个锌指和五个同源结构域组成。作为结构基因组学项目的一部分，我们已经表达了不同人类ZHX基因的单域和多域片段，用于结构测定。利用高通量方法筛选从生物信息学协议中选择的30个单域和多域ZHX1-3构建体，在大肠杆菌中进行可溶性表达。两个同位结构域结晶形成了ZHX1 HD4和ZHX2 HD2的结构。ZHX1 HD4虽然与“homez”和“engrailed”的同源结构域最接近，但表现出结构差异，特别是额外的c端螺旋（螺旋V）缠绕在螺旋I上，从而形成广泛的接触。虽然成功表达和纯化了ZHX2 HD2-3，但在结晶过程中发生了蛋白水解，只产生了HD2晶体。ZHX2 HD2的结构呈现出不寻常的开放构象，螺旋I经历了“结构域交换”形成了一个同型二聚体。虽然生物信息学研究选择的ZHX1的多结构域结构体可以被可溶性表达，但只有单个同源结构域产生晶体。相对于许多已知的同位结构域，ZHX1 HD4的晶体结构表现出额外的疏水相互作用，通过新型c端螺旋V与螺旋i形成广泛的接触，此外，一些带电的协变残基（通常在非等温环境中观察到形成盐桥，如果蝇“嵌入”的同位结构域）被极性残基取代，进一步增加了ZHX1 HD4内部的疏水接触，并潜在地提高了稳定性。相对于纠缠自域。ZHX1 HD4螺旋V点远离正常观察到的DNA主槽结合位点，因此不会阻碍假定的核酸结合。相比之下，对于ZHX2 HD2，观察到的构象改变涉及螺旋I的重排，相对于规范的同源结构域折叠，破坏了正常的DNA结合位点，尽管在同型二聚体形成中观察到蛋白质-蛋白质结合是可能的。
Zhx1 to 3 (zinc-fingers and homeoboxes) form a set of paralogous genes encoding multi-domain proteins. ZHX proteins consist of two zinc fingers followed by five homeodomains. ZHXs have biological roles in cell cycle control by acting as co-repressors of the transcriptional regulator Nuclear Factor Y. As part of a structural genomics project we have expressed single and multi-domain fragments of the different human ZHX genes for use in structure determination. A total of 30 single and multiple domain ZHX1-3 constructs selected from bioinformatics protocols were screened for soluble expression in E. coli using high throughput methodologies. Two homeodomains were crystallized leading to structures for ZHX1 HD4 and ZHX2 HD2. ZHX1 HD4, although closest matched to homeodomains from 'homez' and 'engrailed', showed structural differences, notably an additional C-terminal helix (helix V) which wrapped over helix I thereby making extensive contacts. Although ZHX2 HD2-3 was successfully expressed and purified, proteolysis occurred during crystallization yielding crystals of just HD2. The structure of ZHX2 HD2 showed an unusual open conformation with helix I undergoing 'domain-swapping' to form a homodimer. Although multiple-domain constructs of ZHX1 selected by bioinformatics studies could be expressed solubly, only single homeodomains yielded crystals. The crystal structure of ZHX1 HD4 showed additional hydrophobic interactions relative to many known homeodomains via extensive contacts formed by the novel C-terminal helix V with, in particular, helix I. Additionally, the replacement of some charged covariant residues (which are commonly observed to form salt bridges in non-homeotherms such as the Drosophila 'engrailed' homeodomain), by apolar residues further increases hydrophobic contacts within ZHX1 HD4, and potentially stability, relative to engrailed homeodomain. ZHX1 HD4 helix V points away from the normally observed DNA major groove binding site on homeodomains and thus would not obstruct the putative binding of nucleic acid. In contrast, for ZHX2 HD2 the observed altered conformation involving rearrangement of helix I, relative to the canonical homeodomain fold, disrupts the normal DNA binding site, although protein-protein binding is possible as observed in homodimer formation.
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DOI：
10.1107/s0907444906031337
发表时间：
2006-10-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Berrow, Nick S.;Buessow, K.;Busso, Didier
通讯作者：
Busso, Didier
影响因子：
2.9
作者：
Grant, RA;Rould, MA;Pabo, CO
通讯作者：
Pabo, CO
DOI：
10.1107/s0907444998003254
发表时间：
1998-09-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Brunger, AT;Adams, PD;Warren, GL
通讯作者：
Warren, GL
DOI：
10.1107/s0907444996001783
发表时间：
1996-07-01
期刊：
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
影响因子：
--
作者：
Kleywegt, GJ;Jones, TA
通讯作者：
Jones, TA
影响因子：
7.8
作者：
Chugh, Sumant S.
通讯作者：
Chugh, Sumant S.