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

doi:10.1073/pnas.0802177105

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

*Astbury Centre for Structural Molecular Biology and Institute of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom; and
^†Institute of Structural Molecular Biology, University College London/Birkbeck and School of Crystallography, Malet Street, London WC1E 7HX, United Kingdom

Edited by Arthur Horwich, Yale University School of Medicine, New Haven, CT, and approved July 1, 2008 (received for review March 4, 2008)

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 β-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.

Footnotes

^‡To whom correspondence may be addressed. E-mail: s.e.radford{at}leeds.ac.uk, a.e.ashcroft{at}leeds.ac.uk, or g.waksman{at}ucl.ac.uk
Author contributions: R.J.R., H.R., G.W., A.E.A., and S.E.R. designed research; R.J.R., D.V., T.D., and H.R. performed research; D.V., T.D., and E.P. contributed new reagents/analytic tools; R.J.R. analyzed data; and R.J.R., D.V., T.D., H.R., E.P., G.W., A.E.A., and S.E.R. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0802177105/DCSupplemental.
Freely available online through the PNAS open access option.