HUMAN PLASMA PLATELET ACTIVATING FACTOR ACETYLHYDROLASE

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The plasma form of human platelet activating factor acetylhydrolase (pPAFAH) functions by reducing PAF levels as a general anti-inflammatory scavenger and is linked to anaphylactic shock, asthma and allergic reactions. As a membrane associated protein with no known homologues, pPAFAH is a worthy structural target. Native enzyme has been crystallized in various conditions with a variety of detergents. We currently have high quality crystals (60-100 microns) of active protein (Fig. 1, attached). The best crystals so far have diffracted to a resolution of 3.2 A on our in-house instrument (Fig. 2) with a mosaicity better than 0.7 degrees. The active protein also crystallized in ammonium sulfate and diffracted to a resolution of about 3.5 A, but this crystal (100-120 microns) was highly mosaic. In an alternate approach, we have crystallized a complex of the enzyme with the active site directed inactivator paraoxon, where its active site serine is covalently modified. These crystals (Fig. 3) of inactivated protein diffracted to about 3.5-4.0 A (Fig. 4) but were likewise highly mosaic. Ultimately we would like to pursue both crystal forms, as it is very important to know the structures of both ligand free and active site bound forms of the enzyme. Since pPAFAH does not have a homologous structure, we would like to collect MAD data in order to solve the phases for our structure. Reports suggest Br soaks of proteins are suitable for MAD phasing. Recently a bromide soaked crystal of pPAFAH was screened during the remainder of our lab beam time in August at the APS beamline 14-ID-B. As shown in Fig. 5, this crystal showed clean diffraction to a resolution of 3.4 A and did not suffer appreciable X-ray damage during the 30 min of X-ray exposure. Our preliminary results together with an abundant supply of fresh crystals makes us confident that we will be able to obtain high quality X-ray diffraction for MAD phasing and subsequantly solving the native structure to high resolution.