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Role of conserved salt bridges in homeodomain stability and DNA binding.

ABSTRACT: 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.

SUBMITTER: Torrado M

PROVIDER: S-EPMC2749150 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Role of conserved salt bridges in homeodomain stability and DNA binding.

Torrado Mario M Revuelta Julia J Gonzalez Carlos C Corzana Francisco F Bastida Agatha A Asensio Juan Luis JL

The Journal of biological chemistry 20090626 35

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 positio ...[more]

PMID: 19561080

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Project description:Protein thermal stability is an important factor considered in medical and industrial applications. Many structural characteristics related to protein thermal stability have been elucidated, and increasing salt bridges is considered as one of the most efficient strategies to increase protein thermal stability. However, the accurate simulation of salt bridges remains difficult. In this study, a novel method for salt-bridge design was proposed based on the statistical analysis of 10,556 surface salt bridges on 6,493 X-ray protein structures. These salt bridges were first categorized based on pairing residues, secondary structure locations, and Cα-Cα distances. Pairing preferences generalized from statistical analysis were used to construct a salt-bridge pair index and utilized in a weighted electrostatic attraction model to find the effective pairings for designing salt bridges. The model was also coupled with B-factor, weighted contact number, relative solvent accessibility, and conservation prescreening to determine the residues appropriate for the thermal adaptive design of salt bridges. According to our method, eight putative salt-bridges were designed on a mesophilic β-glucosidase and 24 variants were constructed to verify the predictions. Six putative salt-bridges leaded to the increase of the enzyme thermal stability. A significant increase in melting temperature of 8.8, 4.8, 3.7, 1.3, 1.2, and 0.7°C of the putative salt-bridges N437K-D49, E96R-D28, E96K-D28, S440K-E70, T231K-D388, and Q277E-D282 was detected, respectively. Reversing the polarity of T231K-D388 to T231D-D388K resulted in a further increase in melting temperatures by 3.6°C, which may be caused by the transformation of an intra-subunit electrostatic interaction into an inter-subunit one depending on the local environment. The combination of the thermostable variants (N437K, E96R, T231D and D388K) generated a melting temperature increase of 15.7°C. Thus, this study demonstrated a novel method for the thermal adaptive design of salt bridges through inference of suitable positions and substitutions.

Project description:Revealing the thermodynamic driving force of protein-DNA interactions is crucial to the understanding of factors that dictate the properties and function of protein-DNA complexes. For the binding of DNA to DNA-wrapping proteins, such as the integration host factor (IHF), Record and co-workers proposed that the disruption of a large number of preexisting salt bridges is coupled with the binding process [Holbrook, J. A., et al. (2001) J. Mol. Biol. 310, 379]. To test this proposal, we have conducted explicit solvent MD simulations (multiple ?25-50 ns trajectories for each salt concentration) to examine the behavior of charged residues in IHF, especially concerning their ability to form salt bridges at different salt concentrations. Of the 17 cationic residues noted by Record and co-workers, most are engaged in salt bridge interactions for a significant portion of the trajectories, especially in the absence of salt. This observation suggests that, from a structural point of view, their proposal is plausible. However, the complex behaviors of charged residues observed in the MD simulations also suggest that the unusual thermodynamic characteristics of IHF-DNA binding likely arise from the interplay between complex dynamics of charged residues both in and beyond the DNA binding site. Moreover, a comparison of MD simulations at different salt concentrations suggests that the strong dependence of the IHF-DNA binding enthalpy on salt concentration may not be due to a significant decrease in the number of stable salt bridges in apo IHF at high salt concentrations. In addition to the Hofmeister effects quantified in more recent studies of IHF-DNA binding, we recommend consideration of the variation of the enthalpy change of salt bridge disruption at different salt concentrations. Finally, the simulation study presented here explicitly highlights the fact that the electrostatic properties of DNA-binding proteins can be rather different in the apo and DNA-bound states, which has important implications for the design of robust methods for predicting DNA binding sites in proteins.

Dataset Information

Role of conserved salt bridges in homeodomain stability and DNA binding.

Publications

Role of conserved salt bridges in homeodomain stability and DNA binding.

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