The recruitment of chromatin-modifying coregulator complexes by transcription factors to specific sites of the genome constitutes an important step in many eukaryotic transcriptional regulatory pathways. The histone deacetylase-associated Sin3 corepressor complex is recruited by a large and diverse array of transcription factors through direct interactions with the N-terminal PAH domains of Sin3. Here, we describe the solution structures of the mSin3A PAH1 domain in the apo form and when bound to SAP25, a component of the corepressor complex. Unlike the apo-mSin3A PAH2 domain, the apo-PAH1 domain is conformationally pure and is largely, but not completely, folded. Portions of the interacting segments of both mSin3A PAH1 and SAP25 undergo folding upon complex formation. SAP25 binds through an amphipathic helix to a predominantly hydrophobic cleft on the surface of PAH1. Remarkably, the orientation of the helix is reversed compared to that adopted by NRSF, a transcription factor unrelated to SAP25, upon binding to the mSin3B PAH1 domain. The reversal in helical orientations is correlated with a reversal in the underlying PAH1-interaction motifs, echoing a theme previously described for the mSin3A PAH2 domain. The definition of these so-called type I and type II PAH1-interaction motifs has allowed us to predict the precise location of these motifs within previously experimentally characterized PAH1 binders. Finally, we explore the specificity determinants of protein-protein interactions involving the PAH1 and PAH2 domains. These studies reveal that even conservative replacements of PAH2 residues with equivalent PAH1 residues are sufficient to alter the affinity and specificity of these protein-protein interactions dramatically.

Proteins from thermophilic organisms are able to function under conditions that render a typical mesophilic protein inactive. Pairwise comparisons of homologous mesophilic and thermophilic proteins can help to identify the energetic features of a protein’s energy landscape that lead to such thermostability. Previous studies of bacterial ribonucleases H (RNases H) from the thermophile Thermus thermophilus and the mesophile Escherichia coli revealed that the thermostability arises in part from an unusually low change in heat capacity upon unfolding (ΔCp) for the thermophilic protein. Here, we have further examined how nearly identical proteins can adapt to different thermal constraints by adding a moderately thermophilic homologue to the previously characterized mesophilic and thermophilic pair. We identified a putative RNase H from Chlorobium. tepidum and demonstrated that it is an active RNase H and adopts the RNase H fold. The moderately thermophilic protein has a melting temperature (Tm) similar to that of the mesophilic homologue yet also has a surprisingly low ΔCp, like the thermophilic homologue. This new RNase H folds through a pathway similar to that of the previously studied RNases H. These results suggest that lowering the ΔCp may be a general strategy for achieving thermophilicity for some protein families and implicate the folding core as the major contributor to this effect. It should now be possible to design RNases H that display the desired thermophilic or mesophilic properties, as defined by their ΔCp values, and therefore fine-tune the energy landscape in a predictable fashion.

Ribonucleases H from organisms that grow at different temperatures demonstrate a variable change in heat capacity upon unfolding (ΔC°P) [Ratcliff, K., et al. (2009) Biochemistry 48, 5890-5898]. This ΔC°P has been shown to correlate with a tolerance to higher temperatures and residual structure in the unfolded state of the thermophilic proteins. In the RNase H from Thermus thermophilus, the low ΔC°P has been shown to arise from the same region as the folding core of the protein, and mutagenic studies have shown that loss of a hydrophobic residue in this region can disrupt this residual unfolded state structure and result in a return to a more mesophile-like ΔC°P [Robic, S., et al. (2002) Protein Sci. 11, 381-389;Robic, S., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 11345-11349]. To understand further how residual structure in the unfolded state is encoded in the sequences of these thermophilic proteins, we subjected the RNase H from Chlorobium tepidum to similar studies. Analysis of new chimeric proteins reveals that like T. thermophilus RNase H, the folding core of C. tepidum RNaseH plays an important role in the unfolded state of this protein. Mutagenesis studies, based on both a computational investigation of the hydrophobic networks in the core region and comparisons with similar studies on T. thermophilus RNase H, identify new residues involved in this residual structure and suggest that the residual structure in the unfolded state of C. tepidum RNase H is more restricted than that of T. thermophilus. We conclude that while the folding core region determines the thermophilic-like behavior of this family of proteins, the residue-specific details vary.The mechanisms by which proteins from thermophilic organisms are able to function at extreme temperatures are not well understood. Identifying a more complete set of the rules governing this thermophilicity would further our understanding of proteins from thermophilic † Research in the laboratory of S.M. is supported by Grant GM50945 from the National Institutes of Health.* To whom correspondence should be addressed. Tel: (510) . marqusee@berkeley.edu. SUPPORTING INFORMATION AVAILABLEExamples of standard RNase H activity assays on the RNase H variants CCEO, CCTO, CtepRNH* L56A, and CtepRNH* L56D and raw data from sample denaturant melts used to create the reported protein stability curves. This material is available free of charge via the Internet at http://pubs.acs.org. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2010 July 13. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript organisms and would contribute to our general knowledge about protein stability and energetics. Studies on sets of homologous proteins from thermophilic and mesophilic organisms show that proteins with the same function have nearly identical folds. Furthermore, the sequence and structural similarities between such homologous proteins are often indistinguishable from those between a pair of homologous proteins...

The high oxygen (O 2) sensitivity of green algal [FeFe]-hydrogenases is a significant limitation for the sustained production of hydrogen gas (H 2) from photosynthetic water splitting. To address this limitation we replaced the native [FeFe]-hydrogenases with a more O 2-tolerant clostridial [FeFe]-hydrogenase CaI in Chlamydomonas reinhardtii strain D66ΔHYD (hydA1-hydA2-) that contains insertionally inactivated [FeFe]-hydrogenases genes. Expression and translocation of CaI in D66ΔHYD led to the recovery of H 2 photoproduction at ~20% of the rates of the wild-type parent strain D66. We show for the first time that a bacterial [FeFe]-hydrogenase can be expressed, localized and matured to a catalytically active form that couples to photosynthetic electron transport in the green alga C. reinhardtii. The lower rates of O 2 inactivation of CaI led to more sustained H 2 photoproduction when cultures were challenged with O 2 or kept under prolonged illumination at solar intensities. These results provide new insights into the requisites for attaining photobiological H 2 production from water splitting using a more O 2-tolerant hydrogenase.