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Genetic selection reveals the role of a buried, conserved polar residue.

ABSTRACT: The burial of nonpolar surface area is known to enhance markedly the conformational stability of proteins. The contribution from the burial of polar surface area is less clear. Here, we report on the tolerance to substitution of Ser75 of bovine pancreatic ribonuclease (RNase A), a residue that has the unusual attributes of being buried, conserved, and polar. To identify variants that retain biological function, we used a genetic selection based on the intrinsic cytotoxicity of ribonucleolytic activity. Cell growth at 30 degrees C, 37 degrees C, and 44 degrees C correlated with residue size, indicating that the primary attribute of Ser75 is its small size. The side-chain hydroxyl group of Ser75 forms a hydrogen bond with a main-chain nitrogen. The conformational stability of the S75A variant, which lacks this hydrogen bond, was diminished by DeltaDeltaG = 2.5 kcal/mol. Threonine, which can reinstate this hydrogen bond, provided a catalytically active RNase A variant at higher temperatures than did some smaller residues (including aspartate), indicating that a secondary attribute of Ser75 is the ability of its uncharged side chain to accept a hydrogen bond. These results provide insight on the imperatives for the conservation of a buried polar residue.

SUBMITTER: Johnson RJ

PROVIDER: S-EPMC2203362 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Genetic selection reveals the role of a buried, conserved polar residue.

Johnson R Jeremy RJ Lin Shawn R SR Raines Ronald T RT

Protein science : a publication of the Protein Society 20070801 8

The burial of nonpolar surface area is known to enhance markedly the conformational stability of proteins. The contribution from the burial of polar surface area is less clear. Here, we report on the tolerance to substitution of Ser75 of bovine pancreatic ribonuclease (RNase A), a residue that has the unusual attributes of being buried, conserved, and polar. To identify variants that retain biological function, we used a genetic selection based on the intrinsic cytotoxicity of ribonucleolytic ac ...[more]

PMID: 17656580

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Project description:Strigolactones (SLs) are a class of important plant hormones mainly regulating plant architecture such as branching, which is crucial for crop yield. It is valuable to study SL signaling pathway and its physiological function in sugarcane, the most important sugar crop, for further molecular breeding. Here, two putative SL receptors SsD14a/b and the interacting F-box protein SsMAX2 were identified in Saccharum spontaneum. SL induced both SsD14a and SsD14b to interact with SsMAX2 in yeast. SsD14a, but not SsD14b, could bind with AtMAX2 and AtSMXL7/SsSMXL7. Overexpression of SsD14a or SsMAX2 rescued the increased branching phenotypes of Arabidopsis thaliana d14-1 or max2-3 mutants, respectively. Moreover, the crystal structure of N-terminal truncated SsD14a was solved, with an overall structure identical to AtD14 and OsD14 in the open state, consistent with its conserved branching suppression capacity in Arabidopsis. In line with the biochemical observations, SsD14b could not completely complement in d14-1 although these two SsD14 proteins have almost identical primary sequences except for very few residues. Complement with the combination of SsD14b and SsMAX2 still failed to rescue the d14-1 max2-3 double mutant multi-branching phenotype, indicating SsD14b-AtSMXL7 complex formation is required for regulating branching. Mutagenesis analyses revealed that residue R310 at α10 helix of SsD14a was crucial for the binding with SsSMXL7/AtSMXL7 but not SsMAX2. The site-equivalent single-residue P304R substitution enabled SsD14b to bind with AtMAX2 and AtSMXL7/SsSMXL7 and to rescue the phenotype of d14-1 max2-3 together with SsMAX2. Moreover, this conserved Arg residue across species including rice and Arabidopsis determined the activity of SL receptors through maintaining their interaction with SMXL repressors. Taken together, our work identified conserved and divergent strigolactone receptors in sugarcane core SL signaling pathway and revealed a key residue crucial for plant branching control.

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Genetic selection reveals the role of a buried, conserved polar residue.

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Genetic selection reveals the role of a buried, conserved polar residue.

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