Creating a Homeodomain with High Stability and DNA Binding Affinity by Sequence Averaging

Abstract

There is considerable interest in generating proteins with both high stability and high activity for biomedical and industrial purposes. One approach that has been used successfully to increase the stability of linear repeat proteins is consensus design. It is unclear the extent over which the consensus design approach can be used to produce folded and hyperstable proteins, and importantly, whether such stabilized proteins would retain function. Here we extend the consensus strategy to design a globular protein. We show that a consensus-designed homeodomain (HD) sequence adopts a cooperatively folded homeodomain structure. The unfolding free energy of the consensus-HD is 5 kcal·mol−1 higher than that of the naturally-occurring engrailed-HD from Drosophila melanogaster. Remarkably, the consensus-HD binds the engrailed-HD cognate DNA in a similar mode as the engrailed-HD with approximately 100-fold higher affinity. 15N relaxation studies show a decrease in psec-nsec backbone dynamics in the free state of consensus-HD, suggesting that increased affinity is not a result of increased plasticity. In addition to demonstrating the potential for consensus design of globular proteins with increased stability, these results demonstrate that greatly stabilized proteins can bind cognate substrates with increased affinities, showing that high stability is compatible with function.

Consensus sequence design as a general strategy to create hyperstable, biologically active proteins

“…In both this study and our previous investigations of a consensus homeodomain (30), consensus substitution showed varying effects on molecular recognition.…”

“…We limited our search to folding free energy values determined by chemical denaturation experiments and to proteins that appear to be monomeric ‡ . For comparison, we have included the consensus homeodomain that we recently characterized with this approach (30).…”

“…We compared various sequence properties our six consensus sequences to those of the naturally-occurring sequences within our curated multiple sequence alignments. As with the stability comparisons above, we have included a consensus homeodomain (consensus HD) that we have described previously (30). For every sequence (consensus and every sequence in each MSA), we calculated the proportion of sequence made up of charged residues, polar noncharged residues, total polar residues (the sum of charged and polar noncharged), and nonpolar residues.…”

“…activity are variable. In a recent study, our lab characterized a consensus-designed homeodomain sequence that showed a large increase in both thermodynamic stability and DNA-binding affinity (30). Unlike the point-substitution approach, where both stabilizing and destabilizing substitutions are reported, the success rate of the wholesale approach is not easy to determine from the literature, where publications present single cases of success, whereas failures are not likely to be published.…”

Extreme stability in de novo-designed repeat arrays is determined by unusually stable short-range interactions

“…We note that the fraction of charged residues in the DHR sequences is significantly higher (with an average of 0.45, SI Appendix, Table S1) than the average for all proteins in SWISS-PROT (0.23). An increase in the number of charged residues has been proposed as a mechanism for increased stability in thermophilic proteins (18) and has recently been seen to correlate with high stability in consensus proteins (19).…”