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Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry.

ABSTRACT: P pili are multisubunit fibers essential for the attachment of uropathogenic Escherichia coli to the kidney. These fibers are formed by the noncovalent assembly of six different homologous subunit types in an array that is strictly defined in terms of both the number and order of each subunit type. Assembly occurs through a mechanism termed "donor-strand exchange (DSE)" in which an N-terminal extension (Nte) of one subunit donates a beta-strand to an adjacent subunit, completing its Ig fold. Despite structural determination of the different subunits, the mechanism determining specificity of subunit ordering in pilus assembly remained unclear. Here, we have used noncovalent mass spectrometry to monitor DSE between all 30 possible pairs of P pilus subunits and their Ntes. We demonstrate a striking correlation between the natural order of subunits in pili and their ability to undergo DSE in vitro. The results reveal insights into the molecular mechanism by which subunit ordering during the assembly of this complex is achieved.

SUBMITTER: Rose RJ

PROVIDER: S-EPMC2525559 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Assembly of multi enzyme complexes at subcellular localizations by anchoring- and scaffolding proteins represents a pivotal mechanism for achieving spatiotemporal regulation of cellular signaling after hormone receptor targeting [for review, see (1)]. In the 3' 5'-cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) signaling pathway it is generally accepted that specificity is secured at several levels. This includes at the first level stimulation of receptors coupled to heterotrimeric G proteins which through stimulation of adenylyl cyclase (AC) forms the second messenger cAMP. Cyclic AMP has several receptors including PKA. PKA is a tetrameric holoenzyme consisting of a regulatory (R) subunit dimer and two catalytic (C) subunits. The R subunit is the receptor for cAMP and compartmentalizes cAMP signals through binding to cell and tissue-specifically expressed A kinase anchoring proteins (AKAPs). The current dogma tells that in the presence of cAMP, PKA dissociates into an R subunit dimer and two C subunits which are free to phosphorylate relevant substrates in the cytosol and nucleus. The release of the C subunit has raised the question how specificity of the cAMP and PKA signaling pathway is maintained when the C subunit no longer is attached to the R subunit-AKAP complex. An increasing body of evidence points toward a regulatory role of the cAMP and PKA signaling pathway by targeting the C subunits to various C subunit binding proteins in the cytosol and nucleus. Moreover, recent identification of isoform specific amino acid sequences, motifs and three dimensional structures have together provided new insight into how PKA at the level of the C subunit may act in a highly isoform-specific fashion. Here we discuss recent understanding of specificity of the cAMP and PKA signaling pathway based on C subunit subcellular targeting as well as evolution of the C subunit structure that may contribute to the dynamic regulation of C subunit activity.

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Unraveling the molecular basis of subunit specificity in P pilus assembly by mass spectrometry.

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