Crystal structure and functional studies reveal that PAS factor from Vibrio vulnificus is a novel member of the saposin-fold family.
PAS factor is a novel putative bacterial secretion factor thought to induce secretion of periplasmic proteins. We solved the crystal structure of PAS factor from Vibrio vulnificus at 1.8A resolution and found it to be comprised of five alpha helices that form an antiparallel bundle with an up-and-down topology, and to adopt the saposin-fold characteristic of a family of proteins that bind to membranes and lipids. PAS factor lacks the disulfide bridge characteristic of mammalian saposin-fold proteins; in fact, it shows no sequence homology with mammalian proteins. Nevertheless, the molecular architectures are similar, and the shared propensity for membrane interaction suggests strongly that PAS factor is another member of the saposin-fold family. Analysis of the CD spectra showed that PAS factor binds to membranes directly, while measurement of calcein dye leakage showed that PAS factor interacts strongly with liposomes composed of anionic phospholipids, making them leaky, but binds very weakly with liposomes composed of zwitterionic phospholipids. Moreover, by analyzing tryptophan fluorescence emission from four single-tryptophan mutants (V10W, T22W, F35W, and L70W), we identified the putative phospholipid-binding site of PAS factor. The resultant membrane destabilization likely mediates secretion of periplasmic proteins required for the in vivo survival and pathogenesis of V.vulnificus.
Lee
Jun Hyuck
JH
Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, South Korea.
Yang
Sung-Tae
ST
Rho
Seong-Hwan
SH
Im
Young Jun
YJ
Kim
Soo Young
SY
Kim
Young Ran
YR
Kim
Mun-Kyoung
MK
Kang
Gil Bu
GB
Kim
Jae Il
JI
Rhee
Joon Haeng
JH
Eom
Soo Hyun
SH
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D005452:Fluoresceins / Q000737:chemistry; D005453:Fluorescence; D005456:Fluorescent Dyes; D008081:Liposomes / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010743:Phospholipids / Q000737:chemistry; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D049231:Saposins / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D041261:Vibrio vulnificus / Q000737:chemistry*
D001426:Bacterial Proteins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008081:Liposomes; D010743:Phospholipids; D011994:Recombinant Proteins; D049231:Saposins; D014364:Tryptophan; C007740:fluorexon