Covariation of residues in the homeodomain sequence family

Clarke, Neil D.

doi:10.1002/pro.5560041104

Cited by 94 publications

(74 citation statements)

References 16 publications

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“…Thus, as a first step, the hydrophobic pair Val 19 /Ile 30 , present in the wild-type polypeptide, was replaced by salt bridges of different polarities, either isolated or networked. A previous statistical analysis of the information stored in the homeodomain resource data base (8,9) revealed an intriguing correlation between the nature of pair 19/30 and the loop residue 26 (proline or a branched aliphatic amino acid in all cases). Considering this, both salt bridges and aliphatic residues connecting 19/30 were also considered in the context of either proline or leucine in 26.…”

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Role of Conserved Salt Bridges in Homeodomain Stability and DNA Binding

Torrado

Revuelta

González

et al. 2009

Journal of Biological Chemistry

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The sequence information available for homeodomains reveals that salt bridges connecting pairs 19/30, 31/42, and 17/52 are frequent, whereas aliphatic residues at these sites are rare and mainly restricted to proteins from homeotherms. We have analyzed the influence of salt and hydrophobic bridges at these sites on the stability and DNA binding properties of human Hesx-1 homeodomain. Regarding the protein stability, our analysis shows that hydrophobic side chains are clearly preferred at positions 19/30 and 31/42. This stabilizing influence results from the more favorable packing of the aliphatic side chains with the protein core, as illustrated by the three-dimensional solution structure of a thermostable variant, herein reported. In contrast only polar side chains seem to be tolerated at positions 17/52. Interestingly, despite the significant influence of pairs 19/30 and 31/42 on the stability of the homeodomain, their effect on DNA binding ranges from modest to negligible. The observed lack of correlation between binding strength and conformational stability in the analyzed variants suggests that salt/hydrophobic bridges at these specific positions might have been employed by evolution to independently modulate both properties.Homeodomain proteins are transcription factors present in all eukaryotes and play key roles in cellular differentiation during development (1, 2). The homeodomain encodes a 60-residue DNA binding domain composed of a disordered N-terminal arm, three helical segments (herein referred as I, II, and III) connected by short loops, and a disordered C-terminal region. The secondary and tertiary structures are stabilized by the formation of a well defined hydrophobic core, which results from the packing of the three helices (3, 4).Presently, the largest searchable collection of information for the homeodomain protein family is the Homeodomain Resource Data Base (5-7). It contains around 1056 full-length protein sequences isolated from 112 different species. Inspection of these data shows that helices I/II, I/III, and II/III can be connected by three salt bridges involving pairs 19/30, 31/42, and 17/52, respectively, which in addition can exhibit different polarities. In contrast, hydrophobic bridges at these sites are present only in a minor fraction of sequences (and mainly restricted to homeodomains from warm-blooded animals). This predominance of salt versus hydrophobic bridges suggests a role for the former in homeodomain function and/or stability.Here we analyze the relative effect of salt and hydrophobic bridges on the homeodomain stability and DNA binding properties, employing the human Hesx-1 DNA binding domain as model system. Our approach involves the extensive use of sitedirected mutagenesis experiments together with CD, NMR, and isothermal titration microcalorimetry (ITC) 4 measurements. Thus, as a first step, the hydrophobic pair Val 19 /Ile 30 , present in the wild-type polypeptide, was replaced by salt bridges of different polarities, either isolated or networked. A previous sta...

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confidence: 99%

Role of Conserved Salt Bridges in Homeodomain Stability and DNA Binding

Torrado

Revuelta

González

et al. 2009

Journal of Biological Chemistry

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“…In a covariance analysis of 60 homeodomain protein sequences, Clarke (1995) has observed that residue pairs 19/30. 3 1/42, and 17/52 are among the most strongly correlated covariant residue pairs.…”

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confidence: 99%

“…3 1/42, and 17/52 are among the most strongly correlated covariant residue pairs. Moreover, the nature of these covariances generally functions to preserve salt bridges at these three surface sites (Clarke, 1995). The energetic basis of the requirement for these surface salt bridges is not yet certain.…”

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confidence: 99%

“…The energetic basis of the requirement for these surface salt bridges is not yet certain. Residues Arg 3 1 of engrailed and Arg 42 of Mata2 each make contacts with corresponding phosphate atoms in these homeodomain-DNA complexes, suggesting that there may be a functional constraint requiring a basic residue at one (but not both) of these two sites (Clarke, 1995). However, residue pairs 19/30 and 17/52 are distant from the DNA-binding site and do not interact with DNA in the structures of complexes.…”

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confidence: 99%

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Homology modeling using simulated annealing of restrained molecular dynamics and conformational search calculations with CONGEN: Application in predicting the three‐dimensional structure of murine homeodomain Msx‐1

Tejero

Monleón

et al. 1997

Protein Science

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We have developed an automatic approach for homology modeling using restrained molecular dynamics and simulated annealing procedures, together with conformational search algorithms available in the molecular mechanics program CONGEN (Bruccoleri RE, Karplus M, 1987, Biopolymers 26137-168). The accuracy of the method is validated by "predicting" structures of two homeodomain proteins with known three-dimensional structures, and then applied to predict the three-dimensional structure of the homeodomain of the murine Msx-1 transcription factor. Regions of the unknown protein structure that are highly homologous to the known template structure are constrained by "homology distance constraints," whereas the conformations of nonhomologous regions of the unknown protein are defined only by the potential energy function. A full energy function (excluding explicit solvent) is employed to ensure that the calculated structures have good conformational energies and are physically reasonable. As in NMR structure determinations, information on the consistency of the structure prediction is obtained by superposition of the resulting family of protein structures. In this paper, our homology modeling algorithm is described and compared with related homology modeling methods using spatial constraints derived from the structures of homologous proteins. The software is then used to predict the DNA-bound structures of three homeodomain proteins from the X-ray crystal structure of the engrailed homeodomain protein (Kissinger CR et al., 1990, Cell 63:579-590). The resulting backbone and side-chain conformations of the modeled yeast MatcY2 and D. melunogaster Antennapedia homeodomains are excellent matches to the corresponding published X-ray crystal (Wolberger C et al., 1991, Cell 67517-528) and NMR , J Mol Biol234:1084-1097) structures, respectively. Examination of these structures of Msx-1 reveals a network of highly conserved surface salt bridges that are proposed to play a role in regulating protein-protein interactions of homeodomains in transcription complexes. Abbreviations: Antp, homeodomain of the D. melanogusrer Antennapedia protein; DG, distance geometry calculations using metric-mauix embedding methods; Conf. E., total conformational energy including electrostatic effects, computed from the CHARMM potential function; Mata2, homeodomain of the yeast M a t d protein; Msx-1, homeodomain of the murine Msx-1 protein; pdf, probability density function; RMSD, R M S deviation; SARMD, simulated annealing with restrained molecular dynamics; VDW E., van der Waals energy computed from the Lennard-Jones portion of the CHARMM potential function. Keywords 956Homology modeling of homeodomain Msx-1 with CONGEN 957The homeodomain is a highly conserved sequence-specific DNAbinding domain that has been found in many transcription factors. First discovered in Drosophila melanogaster, homeodomains have been found in almost every organism from nematodes to humans (Kessel & Gruss, 1990; Wang et al., 1993), and have been found to play a fu...

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“…A single point mutation might ease or complicate each imaginable path of mutation at any other position in the complex, regardless of its distance from the interface (Lovell and Robertson 2010). In fact, co-evolution events have been detected affecting sites that are distant structurally (Gobel et al 1994;Clarke 1995;Gloor et al 2005;Fares and McNally 2006). On the other hand, the probability of correlated amino acid changes is closely related to the chemical nature of changes.…”

Section: Co-evolution Methodsmentioning

confidence: 99%