The structure of DinB from Geobacillus stearothermophilus: a representative of a unique four-helix-bundle superfamily.

The crystal structure of the dinB gene product from Geobacillus stearothermophilus (GsDinB) is reported at 2.5 A resolution. The dinB gene is one of the DNA-damage-induced genes and the corresponding protein, DinB, is the founding member of a Pfam family with no known function. The protein contains a four-helix up-down-down-up bundle that has previously been described in the literature in three disparate proteins: the enzyme MDMPI (mycothiol-dependent maleylpyruvate isomerase), YfiT and TTHA0303, a member of a small DUF (domain of unknown function). However, a search of the DALI structural database revealed similarities to a further 11 new unpublished structures contributed by structural genomics centers. The sequences of these proteins are quite divergent and represent several Pfam families, yet their structures are quite similar and most (but not all) seem to have the ability to coordinate a metal ion using a conserved histidine-triad motif. The structural similarities of these diverse proteins suggest that a new Pfam clan encompassing the families that share this fold should be created. The proteins that share this fold exhibit four different quaternary structures: monomeric and three different dimeric forms.

Protein crystallization by surface entropy reduction: optimization of the SER strategy.

A strategy of rationally engineering protein surfaces with the aim of obtaining mutants that are distinctly more susceptible to crystallization than the wild-type protein has previously been suggested. The strategy relies on replacing small clusters of two to three surface residues characterized by high conformational entropy with alanines. This surface entropy reduction (or SER) method has proven to be an effective salvage pathway for proteins that are difficult to crystallize. Here, a systematic comparison of the efficacy of using Ala, His, Ser, Thr and Tyr to replace high-entropy residues is reported. A total of 40 mutants were generated and screened using two different procedures. The results reaffirm that alanine is a particularly good choice for a replacement residue and identify tyrosines and threonines as additional candidates that have considerable potential to mediate crystal contacts. The propensity of these mutants to form crystals in alternative screens in which the normal crystallization reservoir solutions were replaced with 1.5 M NaCl was also examined. The results were impressive: more than half of the mutants yielded a larger number of crystals with salt as the reservoir solution. This method greatly increased the variety of conditions that yielded crystals. Taken together, these results suggest a powerful crystallization strategy that combines surface engineering with efficient screening using standard and alternate reservoir solutions.