PAS Domain Allostery and Light-induced Conformational Changes in Photoactive Yellow Protein upon I2 Intermediate Formation, Probed with Enhanced Hydrogen/Deuterium Exchange Mass Spectrometry

Abstract

Photoactive yellow protein (PYP) is a small bacterial photoreceptor that undergoes a light-activated reaction cycle. PYP is also the prototypical Per-Arnt-Sim (PAS) domain. PAS domains, found in diverse multi-domain proteins from bacteria to humans, mediate protein-protein interactions and function as sensors and signal transducers. Here, we investigate conformational and dynamic changes in solution in wild-type PYP upon formation of the long-lived putative signaling intermediate I2 with enhanced hydrogen/deuterium exchange mass spectrometry (DXMS). The DXMS results showed that the central beta-sheet remains stable but specific external protein segments become strongly deprotected. Light-induced disruption of the dark-state hydrogen bonding network in I2 produces increased flexibility and opening of PAS core helices alpha3 and alpha4, releases the beta4-beta5 hairpin, and propagates conformational changes to the central beta-sheet. Surprisingly, the first approximately 10 N-terminal residues, which are essential for fast dark-state recovery from I2, become more protected. By combining the DXMS results with our crystallographic structures, which reveal detailed changes near the chromophore but limited protein conformational change, we propose a mechanism for I2 state formation. This mechanism integrates the results from diverse biophysical studies of PYP, and links an allosteric T to R-state conformational transition to three pathways for signal propagation within the PYP fold. On the basis of the observed changes in PYP plus commonalities shared among PAS domain proteins, we further propose that PAS domains share this conformational mechanism, which explains the versatile signal transduction properties of the structurally conserved PYP/PAS module by framework-encoded allostery.

Calcium Binding Rigidifies the C2 Domain and the Intradomain Interaction of GIVA Phospholipase A2 as Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry

“…Proteolysis Liquid Chromatography-Mass Spectrometry Analysis of Samples-All steps were performed at 0°C as previously described (26,28). The samples were hand-thawed on melting ice and injected onto and passed through a protease column (66-l bed volume) filled with porcine pepsin (Sigma; immobilized on Poros 20 AL medium at 30 mg/ml following the manufacturer's instructions, Applied Biosystems) at a flow rate of 100 l/min with 0.05% trifluoroacetic acid.…”

“…The peptides were eluted at 50 l/min with a linear gradient of 0.046% trifluoroacetic acid, 6.4% (v/v) acetonitrile to 0.03% trifluoroacetic acid, 38.4% acetonitrile for 30 min. The eluate from the C18 column was directed to a Finnigan Classic LCQ mass spectrometer via its ESI probe operated with a capillary temperature of 200°C as previously described (26,28).…”

“…DXMS Explorer (Sierra Analytics Inc, Modesto CA) was used for the analysis of the mass spectra as previously described (26,28). All selected peptides had first passed the quality control threshold of the software and were then manually checked for the mass envelope fitting with the calculated mass envelope for data reduction.…”

“…Peptide amide hydrogen/deuterium exchange coupled with liquid chromatography mass spectrometry (DXMS) has been widely used to analyze the interface of protein-protein interactions (25,26), protein conformational changes (27,28), and protein dynamics (29,30 …”

Hydrogen bond dynamics in the active site of photoactive yellow protein

“…PYP serves as the structural prototype of the Per-Arnt-Sim (PAS) sensory domain and has provided a powerful system to dissect the physical mechanisms of light-activated signal transduction (9)(10)(11). Upon absorption of light at 446 nm and rapid charge rearrangement within its thioester-linked p-coumaric acid chromophore, PYP initiates a complex photocycle that couples the local trans-cis isomerization of a double bond within the chromophore to global conformational restructuring of the protein on the picosecond-to-millisecond time scale (12)(13)(14). The phenolic chromophore is stabilized in the photochemical ground state as a phenolate anion by hydrogen bonds donated by Tyr-42 and protonated Glu-46, which form an extended hydrogen bond network within the active site via additional interactions with the side-chain hydroxyl of Thr-50 and backbone amide of Arg-52 ( Fig.…”