Dissecting interdomain communication within cAPK regulatory subunit type IIβ using enhanced amide hydrogen/deuterium exchange mass spectrometry (DXMS)

Abstract

AbstractcAMP-dependent protein kinase (cAPK) is a heterotetramer containing a regulatory (R) subunit dimer bound to two catalytic (C) subunits and is involved in numerous cell signaling pathways. The C-subunit is activated allosterically when two cAMP molecules bind sequentially to the cAMP-binding domains, designated A and B (cAB-A and cAB-B, respectively). Each cAMP-binding domain contains a conserved Arg residue that is critical for high-affinity cAMP binding. Replacement of this Arg with Lys affects cAMP affinity, the structural integrity of the cAMP-binding domains, and cAPK activation. To better understand the local and long-range effects that the Arg-to-Lys mutation has on the dynamic properties of the R-subunit, the amide hydrogen/deuterium exchange in the RII␤ subunit was probed by electrospray mass spectrometry. Mutant proteins containing the Arg-to-Lys substitution in either cAMP-binding domain were deuterated for various times and then, prior to mass spectrometry analysis, subjected to pepsin digestion to localize the deuterium incorporation. Mutation of this Arg in cAB-A (Arg230) causes an increase in amide hydrogen exchange throughout the mutated domain that is beyond the modest and localized effects of cAMP removal and is indicative of the importance of this Arg in domain organization. Mutation of Arg359 (cAB-B) leads to increased exchange in the adjacent cAB-A domain, particularly in the cAB-A domain C-helix that lies on top of the cAB-B domain and is believed to be functionally linked to the cAB-B domain. This interdomain communication appears to be a unidirectional pathway, as mutation of Arg230 in cAB-A does not effect dynamics of the cAB-B domain.

Rapid refinement of crystallographic protein construct definition employing enhanced hydrogen/deuterium exchange MS

“…Thirty microliters of a stock exchange quench solution [0.8% formic acid, 1.6 M guanidine hydrochloride (Gdn⅐HCl) was then added to each sample (final concentration 0.5% formic acid, 1.0 M Gdn⅐HCl), samples were transferred to autosampler vials, and were then frozen on dry ice within 1 min after addition of quench solution as described (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38). Vials with frozen samples were stored at Ϫ80°C until they were transferred to the dry icecontaining sample basin of the cryogenic autosampler module of a DXMS analysis apparatus designed and operated as described (36)(37)(38). In brief, samples were melted at 0°C, were proteolyzed for 16 sec by exposure to immobilized pepsin, and fragments were collected on a c18 HPLC column, with subsequent acetonitrile gradient elution.…”

“…In brief, samples were melted at 0°C, were proteolyzed for 16 sec by exposure to immobilized pepsin, and fragments were collected on a c18 HPLC column, with subsequent acetonitrile gradient elution. Column effluent was analyzed on both a Thermo Finnigan LCQ electrospray mass spectrometer and a Micromass Q-Tof mass spectrometer, as described (32)(33)(34)(35)(36)(37)(38). The SEQUEST software program (Thermo Finnigan, San Jose, CA) identified the likely sequence of the parent peptide ions and these tentative identifications were confirmed with specialized DXMS data reduction software as described (36)(37)(38).…”

“…Data on the deuterated sample set were acquired in a single automated 30-h run and subsequent data reduction was performed with the DXMS software. Corrections for loss of deuterium label were made as described (36)(37)(38). The total time elapsed for data acquisition and analysis (both fragmentation maps and deuteration study) was 2 weeks.…”

“…This approach optimizes our ability to measure the widely ranging exchange rates for most of the peptide amides in the protein (32)(33)(34)(35)(36)(37)(38). In the present study, we sought to localize only disordered amides that exchanged very fast in the native protein.…”

“…Building on the pioneering work of Englander and coworkers (14,15,27), we have developed and implemented a number of improvements that have significantly improved throughput, comprehensiveness, and resolution. We term the method employing these enhancements high-throughput and highresolution deuterium exchange MS (DXMS) (28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38).…”