A conserved helical capping hydrogen bond in PAS domains controls signaling kinetics in the superfamily prototype photoactive yellow protein.
PAS domains form a divergent protein superfamily with more than 20 000 members that perform a wide array of sensing and regulatory functions in all three domains of life. Only nine residues are well-conserved in PAS domains, with an Asn residue at the start of α-helix 3 showing the strongest conservation. The molecular functions of these nine conserved residues are unknown. We use static and time-resolved visible and FTIR spectroscopy to investigate receptor activation in the photosensor photoactive yellow protein (PYP), a PAS domain prototype. The N43A and N43S mutants allow an investigation of the role of side-chain hydrogen bonding at this conserved position. The mutants exhibit a blue-shifted visible absorbance maximum and up-shifted chromophore pK(a). Disruption of the hydrogen bonds in N43A PYP causes both a reduction in protein stability and a 3400-fold increase in the lifetime of the signaling state of this photoreceptor. A significant part of this increase in lifetime can be attributed to the helical capping interaction of Asn43. This extends the known importance of helical capping for protein structure to regulating functional protein kinetics. A model for PYP activation has been proposed in which side-chain hydrogen bonding of Asn43 is critical for relaying light-induced conformational changes. However, FTIR spectroscopy shows that both Asn43 mutants retain full allosteric transmission of structural changes. Analysis of 30 available high-resolution structures of PAS domains reveals that the side-chain hydrogen bonding of residue 43 but not residue identity is highly conserved and suggests that its helical cap affects signaling kinetics in other PAS domains.
Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, Oklahoma 74078, United States.
Masato
M
Kumauchi
Sandip
S
Kaledhonkar
Andrew F
AF
Philip
James
J
Wycoff
Miwa
M
Hara
Yunxing
Y
Li
Aihua
A
Xie
Wouter D
WD
Hoff
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020283:Photoreceptors, Microbial / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D015398:Signal Transduction; D017550:Spectroscopy, Fourier Transform Infrared
D020283:Photoreceptors, Microbial