Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase

doi:10.1074/jbc.M109.004317

Defining the Structural Basis of Human Plasminogen Binding by Streptococcal Surface Enolase*^♦

From the ^‡School of Biological Sciences and
^§School of Chemistry, University of Wollongong, Wollongong NSW 2522, Australia, the
^¶Department of Genetics of Microorganisms, Institute for Genetics and Functional Genomics, Ernst-Moritz-Arndt University of Greifswald, Greifswald D-17487, Germany, the
^‖School of Molecular and Microbial Sciences and Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia, the
**Department of Pathology, Ohio State University, Columbus, Ohio 43210, and the
^‡‡Department of Chemistry, University of Cambridge, Cambridge CB2 1TN, United Kingdom

⁷ To whom correspondence should be addressed:
School of Biological Sciences, University of Wollongong, Northfields Ave., Wollongong NSW 2522, Australia.
Tel.: 61-2-4221-3439; Fax: 61-2-4221-4135; E-mail: mwalker{at}uow.edu.au.

Abstract

The flesh-eating bacterium group A Streptococcus (GAS) binds and activates human plasminogen, promoting invasive disease. Streptococcal surface enolase (SEN), a glycolytic pathway enzyme, is an identified plasminogen receptor of GAS. Here we used mass spectrometry (MS) to confirm that GAS SEN is octameric, thereby validating in silico modeling based on the crystal structure of Streptococcus pneumoniae α-enolase. Site-directed mutagenesis of surface-located lysine residues (SEN^{K252 + 255A}, SEN^K304A, SEN^K334A, SEN^K344E, SEN^K435L, and SEN^Δ434–435) was used to examine their roles in maintaining structural integrity, enzymatic function, and plasminogen binding. Structural integrity of the GAS SEN octamer was retained for all mutants except SEN^K344E, as determined by circular dichroism spectroscopy and MS. However, ion mobility MS revealed distinct differences in the stability of several mutant octamers in comparison with wild type. Enzymatic analysis indicated that SEN^K344E had lost α-enolase activity, which was also reduced in SEN^K334A and SEN^Δ434–435. Surface plasmon resonance demonstrated that the capacity to bind human plasminogen was abolished in SEN^{K252 + 255A}, SEN^K435L, and SEN^Δ434–435. The lysine residues at positions 252, 255, 434, and 435 therefore play a concerted role in plasminogen acquisition. This study demonstrates the ability of combining in silico structural modeling with ion mobility-MS validation for undertaking functional studies on complex protein structures.

Footnotes

↵¹ A recipient of a University of Wollongong Postgraduate Award.
↵² An Australian Research Council (ARC) Federation Fellow and an NHMRC Honorary Research Fellow.
↵³ A Royal Society University Research Fellow.
↵⁴ A Royal Society Professor.
↵⁵ An ARC Australian Professorial Fellow.
↵⁶ An NHMRC R. D. Wright Fellow.
↵* This work was supported by the National Health and Medical Research Council (NHMRC) of Australia Grant 459103.
↵^♦ This article was selected as a Paper of the Week.
↵ The on-line version of this article (available at http://www.jbc.org) contains a supplemental table and two supplemental figures.
↵8 The abbreviations used are:

GAS

group A Streptococcus

SEN

surface enolase

MS

mass spectrometry

IM

ion mobility

ESI

electrospray mass ionization

Tof

time-of-flight

mbar

millibar.
- Received February 27, 2009.
- Revision received April 5, 2009.