Alignment and structure prediction of divergent protein families: periplasmic and outer membrane proteins of bacterial efflux pumps

Broad-specificity efflux pumps have been implicated in multidrug-resistant strains of Pseudomonas aeruginosa and other Gram-negative bacteria. Most Gram-negative pumps of clinical relevance have three components, an inner membrane transporter, an outer membrane channel protein, and a periplasmic protein, which together coordinate efflux from the cytoplasmic membrane across the outer membrane through an unknown mechanism. The periplasmic efflux proteins (PEPs) and outer membrane efflux proteins (OEPs) are not obviously related to proteins of known structure, and understanding the structure and function of these proteins has been hindered by the difficulty of obtaining reasonable multiple alignments. We present a general strategy for the alignment and structure prediction of protein families with low mutual sequence similarity using the PEP and OEP families as detailed examples. Gibbs sampling, hidden Markov models, and other analysis techniques were used to locate motifs, generate multiple alignments, and assign PEP or OEP function to hypothetical proteins in several species. We also developed an automated procedure which combines multiple alignments with structure prediction algorithms in order to identify conserved structural features in protein families. This process was used to identify a probable alpha-helical hairpin in the PEP family and was applied to the detection of transmembrane beta-strands in OEPs. We also show that all OEPs contain a large tandem duplication, and demonstrate that the OEP family is unlikely to adopt a porin fold, in contrast to previous predictions.