Cargando…

Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains

BACKGROUND: 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 Nucl...

Descripción completa

Detalles Bibliográficos
Autores principales: Bird, Louise E, Ren, Jingshan, Nettleship, Joanne E, Folkers, Gert E, Owens, Raymond J, Stammers, David K
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2893186/
https://www.ncbi.nlm.nih.gov/pubmed/20509910
http://dx.doi.org/10.1186/1472-6807-10-13
_version_ 1782183019759534080
author Bird, Louise E
Ren, Jingshan
Nettleship, Joanne E
Folkers, Gert E
Owens, Raymond J
Stammers, David K
author_facet Bird, Louise E
Ren, Jingshan
Nettleship, Joanne E
Folkers, Gert E
Owens, Raymond J
Stammers, David K
author_sort Bird, Louise E
collection PubMed
description BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.
format Text
id pubmed-2893186
institution National Center for Biotechnology Information
language English
publishDate 2010
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-28931862010-06-29 Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains Bird, Louise E Ren, Jingshan Nettleship, Joanne E Folkers, Gert E Owens, Raymond J Stammers, David K BMC Struct Biol Research article BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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. BioMed Central 2010-05-28 /pmc/articles/PMC2893186/ /pubmed/20509910 http://dx.doi.org/10.1186/1472-6807-10-13 Text en Copyright ©2010 Bird et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research article
Bird, Louise E
Ren, Jingshan
Nettleship, Joanne E
Folkers, Gert E
Owens, Raymond J
Stammers, David K
Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains
title Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains
title_full Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains
title_fullStr Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains
title_full_unstemmed Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains
title_short Novel structural features in two ZHX homeodomains derived from a systematic study of single and multiple domains
title_sort novel structural features in two zhx homeodomains derived from a systematic study of single and multiple domains
topic Research article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2893186/
https://www.ncbi.nlm.nih.gov/pubmed/20509910
http://dx.doi.org/10.1186/1472-6807-10-13
work_keys_str_mv AT birdlouisee novelstructuralfeaturesintwozhxhomeodomainsderivedfromasystematicstudyofsingleandmultipledomains
AT renjingshan novelstructuralfeaturesintwozhxhomeodomainsderivedfromasystematicstudyofsingleandmultipledomains
AT nettleshipjoannee novelstructuralfeaturesintwozhxhomeodomainsderivedfromasystematicstudyofsingleandmultipledomains
AT folkersgerte novelstructuralfeaturesintwozhxhomeodomainsderivedfromasystematicstudyofsingleandmultipledomains
AT owensraymondj novelstructuralfeaturesintwozhxhomeodomainsderivedfromasystematicstudyofsingleandmultipledomains
AT stammersdavidk novelstructuralfeaturesintwozhxhomeodomainsderivedfromasystematicstudyofsingleandmultipledomains