935 Purification and Crystallization of PadA of Streptococcus gordonii

Friday, March 23, 2012: 2 p.m. - 3:15 p.m.
Presentation Type: Poster Session
L. WARREN1, M. LARSON2, S. KHALED2, H. PETERSEN3, S.W. KERRIGAN4, H. JENKINSON3, and C. DEIVANAYAGAM2, 1School of Dentistry, University of Alabama at Birmingham, Birmingham, AL, 2Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL, 3Department of Oral and Dental Sciences, University of Bristol, Bristol, United Kingdom, 4Royal College of Surgeons in Ireland, Dublin, Ireland
Objective: Streptococcus gordonii (S. gordonii) is an oral bacterium implicated in infective endocarditis.  A surface protein of S. gordonii, PadA (platelet adherence domain A), adheres to the platelet integrin GPIIbIIIa.  This interaction is considered a potential target to develop preventive therapies for bacterial endocarditis.  The structure of PadA is currently unknown, and this study aims to determine the atomic resolution structure of PadA using X-ray crystallography.

Method: The N-terminal region of PadA binds to platelets, and, therefore, several N-terminal fragments were cloned and expressed in E. coli BL21 (DE3) cells harboring the appropriate vectors for each fragment. These N-terminal fragments were purified using a three-step protocol.  First, the histidine-tagged proteins were purified over a nickel affinity column, then purified by anionic exchange, and finally polished over size exclusion chromatography.  Highly purified (>99%) proteins were concentrated under nitrogen gas and then screened for crystallization conditions using the hanging drop method.  Several hits were obtained, and these crystallization conditions were further optimized to grow diffraction quality crystals.

Result: Among several fragments of PadA that were highly purified, only one fragment of PadA readily crystallized.  X-ray diffraction data was collected on an RU-H3R generator operating at 50 V and 100 A, fitted with an RAXIS IV image plate detector.  Data collected and scaled indicated that the PadA crystals adopted a trigonal space group (either P321, P3121, or P3221) with the following cell parameters: a = 154.46, b = 154.46, c = 235.62, and α=β= 90 with γ= 120.

Conclusion: The functional region of Streptococcus gordonii PadA implicated in binding to the platelet integrin GPIIbIIIa has been crystallized, and structure solution is in progress.

Support: NIDCR-5T32DE017607 DART

This abstract is based on research that was funded entirely or partially by an outside source: NIDCR-5T32DE017607DART

Keywords: Adherence and colonization, Bacterial, Infection, Plaque and Structure