Proteins that share common ancestry may differ in structure and function because of divergent evolution of their amino acid sequences. For a typical diverse protein superfamily, the properties of a few scattered members are known from experiment. A satisfying picture of functional and structural evolution in relation to sequence changes, however, may require characterization of a larger, well chosen subset. Here, we employ a ''stepping-stone'' method, based on transitive homology, to target sequences intermediate between two related proteins with known divergent properties. We apply the approach to the question of how new protein folds can evolve from preexisting folds and, in particular, to an evolutionary change in secondary structure and oligomeric state in the Cro family of bacteriophage transcription factors, initially identified by sequence-structure comparison of distant homologs from phages P22 and . We report crystal structures of two Cro proteins, Xfaso 1 and Pfl 6, with sequences intermediate between those of P22 and . The domains show 40% sequence identity but differ by switching of ␣-helix to ␤-sheet in a C-terminal region spanning Ϸ25 residues. Sedimentation analysis also suggests a correlation between helix-to-sheet conversion and strengthened dimerization.conformational switching ͉ structural evolution ͉ transitive homology ͉ x-ray crystallography T he amino acid sequences of proteins evolve faster than the structures and functions encoded by these sequences. This neutral sequence drift allows annotation of an uncharacterized protein-coding gene based on common ancestry (homology) with a characterized gene, even if the protein sequences are quite different. Conservation of structure and function may hold even for homology so distant that no clear sequence similarity has survived evolutionary divergence. Yet there are limits: the structural and functional evolution of proteins is not completely static, and the likelihood of two proteins evolving divergent properties increases with the extent of sequence change separating them. Remote homology detection methods [for example, PSI-BLAST (1), COMPASS (2), and HHpred (3)] thus yield diminishing returns for gene annotation by grouping distantly related proteins into superfamilies that encompass diverse properties and biological roles. Simultaneously, however, the excavation of distant relationships opens a rich field for experimental studies of protein evolution, with the promise of recovered annotation power as one elucidates how structure and function vary across the ''sequence space'' of a superfamily.Transitive sequence comparison is one method for detecting distant homology between highly diverged sequences (4-8). In this approach, two dissimilar sequences, A and C, are indirectly linked if a third ''intermediate'' sequence B exists with sufficient similarity to both A and C to imply homology with both proteins. The relationships between A and B and between B and C combine to support distant common ancestry between A and C. Transitivity can extend ...

Caseinolytic peptidases (ClpPs) regulate diverse aspects of cellular physiology in bacteria. Some species have multiple ClpPs, including the opportunistic pathogen Pseudomonas aeruginosa, in which there is an archetypical isoform, ClpP1, and a second isoform, ClpP2, about which little is known. Here, we use phenotypic assays to investigate the biological roles of ClpP1 and ClpP2 and biochemical assays to characterize purified ClpP1, ClpP2, ClpX, and ClpA. Interestingly, ClpP1 and ClpP2 have distinct intracellular roles for motility, pigment production, iron scavenging, and biofilm formation. Of particular interest, ClpP2, but not ClpP1, is required for microcolony organization, where multicellular organized structures first form on the pathway to biofilm production. We found that purified ClpP1 with ClpX or ClpA was enzymatically active, yet to our surprise, ClpP2 was inactive and not fully assembled in vitro; attempts to assist ClpP2 assembly and activation by mixing with the other Clp components failed to turn on ClpP2, as did solution conditions that have helped activate other ClpPs in vitro. We postulate that the active form of ClpP2 has yet to be discovered, and we present several potential models to explain its activation as well as the unique role ClpP2 plays in the development of the clinically important biofilms in P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is responsible for severe infections of immunocompromised patients. Our work demonstrates that two different isoforms of the Clp peptidase, ClpP1 and ClpP2, control distinct aspects of cellular physiology for this organism. In particular, we identify ClpP2 as being necessary for microcolony organization. Pure active forms of ClpP1 and either ClpX or ClpA were characterized as assembled and active, and ClpP2 was incompletely assembled and inactive. By establishing both the unique biological roles of ClpP1 and ClpP2 and their initial biochemical assemblies, we have set the stage for important future work on the structure, function, and biological targets of Clp proteolytic enzymes in this important opportunistic pathogen.

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Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double‐ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen Pseudomonas aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP17P27 tetradecamer that is required for proper biofilm development. PaClpP114 and PaClpP17P27 complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non‐canonical features in its N‐ and C‐terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late‐stage P. aeruginosa development.

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