Structural Characterization of Protein Kinase A as a Function of Nucleotide Binding

Andersen, Mikkel Østerheden; Shaffer, Jennifer; Jennings, Patricia A.; Adams, Joseph A.

doi:10.1074/jbc.m011543200

Cited by 51 publications

(29 citation statements)

References 74 publications

(76 reference statements)

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“…In the single-domain protein interleukin-1β, point mutations that are distal to the receptor binding interface have no effect on stability, yet they have significant effects on the binding properties (55). In multidomain proteins, there are also examples of transdomain communication across individual domains, including in cAMP-dependent protein kinase (56) and in COOH-terminal Src kinase (Csk) (7). It was demonstrated (57) that chemical perturbations are linked to activity regulation through a "communication pathway" between a distal SH2 domain and the active site.…”

Section: Discussionmentioning

confidence: 99%

Allostery in the ferredoxin protein motif does not involve a conformational switch

Nechushtai

Lammert²,

Michaeli³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Regulation of protein function via cracking, or local unfolding and refolding of substructures, is becoming a widely recognized mechanism of functional control. Oftentimes, cracking events are localized to secondary and tertiary structure interactions between domains that control the optimal position for catalysis and/or the formation of protein complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip the balance and provide a regulatory functional switch. However, understanding the factors controlling function in single-domain proteins is still a significant challenge to structural biologists. We investigated the functional landscape of a single-domain planttype ferredoxin protein and the effect of a distal loop on the electron-transfer center. We find the global stability and structure are minimally perturbed with mutation, whereas the functional properties are altered. Specifically, truncating the L1,2 loop does not lead to large-scale changes in the structure, determined via X-ray crystallography. Further, the overall thermal stability of the protein is only marginally perturbed by the mutation. However, even though the mutation is distal to the iron-sulfur cluster (∼20 Å), it leads to a significant change in the redox potential of the ironsulfur cluster (57 mV). Structure-based all-atom simulations indicate correlated dynamical changes between the surface-exposed loop and the iron-sulfur cluster-binding region. Our results suggest intrinsic communication channels within the ferredoxin fold, composed of many short-range interactions, lead to the propagation of long-range signals. Accordingly, protein interface interactions that involve L1,2 could potentially signal functional changes in distal regions, similar to what is observed in other allosteric systems.electron transfer | functional energy landscape | iron-sulfur proteins | protein folding O ver the last several decades, our understanding of protein function has evolved from a rather static perspective, where signaling and function have been understood through surface complementarity arguments, such as the "lock and key" paradigm (1, 2), to a more dynamic view where protein conformational fluctuations are inextricably linked to function (3). As our understanding of protein dynamics expands, we are revealing many mechanisms by which proteins exploit conformational fluctuations to perform cellular function. In multidomain proteins, relative repositioning of domains is often linked to their levels of activity. For example, large-scale domain rearrangements in the four-domain Src kinase (4) and C-terminal Src kinase (5, 6) lead to these proteins being in so-called "on" or "off" states, and functional regulation may be obtained by adjusting the balance between these conformations (7). In proteins such as adenylate kinase, domain rearrangements can be rate limiting during each round of catalysis (8), where the enzyme cycles between ligandcompetent and ligand-release conformations. Because the kinetics of these tra...

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Section: Discussionmentioning

confidence: 99%

Allostery in the ferredoxin protein motif does not involve a conformational switch

Nechushtai

Lammert²,

Michaeli³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…Only residues 113-244 of the R I ␣ subunit were used in the docking, and residues 245-376 were added after the docked solutions were obtained. ATP and the 20-residue peptide PKI (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) were retained in the C-subunit structure. The C-subunit was held stationary while the R-subunit was rotated and translated about it.…”

Section: Methodsmentioning

confidence: 99%

“…Examination of the 15 DOT solutions that survived the distance constraint filters revealed that they all satisfied the interaction between Glu-143 (from R I ␣) and Lys-213 (from C-subunit) required by the experimental result that mutations at Lys-213 of the C-subunit complemented those at Glu-143 in R I ␣ (1, 7). They also all satisfied a distance constraint connecting the pseudosubstrate͞inhibitor sequence (mimicked by the PKI (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24) and the N terminus of the structure of R I ␣ (residue 113). Finally, residues 92-100 in the C-subunit and residues 122-136, 204-221, and 239-244 in R I ␣ that did not show any protection on complex formation were not at the interface in the final model.…”

Section: Computation Of All Possible Docked Structures Of the Pka Holmentioning

confidence: 99%

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Identification of the protein kinase A regulatory R ^I α-catalytic subunit interface by amide H/ ² H exchange and protein docking

Anand

Law

Mandell

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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An important goal after structural genomics is to build up the structures of higher-order protein-protein complexes from structures of the individual subunits. Often structures of higher order complexes are difficult to obtain by crystallography. We have used an alternative approach in which the structures of the individual catalytic (C) subunit and R I ␣ regulatory (R) subunit of PKA were first subjected to computational docking, and the top 100,000 solutions were subsequently filtered based on amide hydrogen͞ deuterium (H͞ 2 H) exchange interface protection data. The resulting set of filtered solutions forms an ensemble of structures in which, besides the inhibitor peptide binding site, a flat interface between the C-terminal lobe of the C-subunit and the A-and B-helices of R I ␣ is uniquely identified. This holoenzyme structure satisfies all previous experimental data on the complex and allows prediction of new contacts between the two subunits.matrix-assisted laser desorption ionization/time-of-flight ͉ protein-protein interactions ͉ PKA holoenzyme (type I) P rotein kinase A (PKA), a central locus for cAMP-mediated signaling in mammalian cells, exists in an inactive state as a tetrameric holoenzyme composed of two regulatory (R) and two catalytic (C) subunits. Binding of cAMP to the R-subunits leads to dissociation of the holoenzyme and unleashing of the active C-subunit. Like many protein kinases, inhibition of the Csubunit by the R I ␣ regulatory subunit occurs by bipartite binding to the C-subunit. The R-subunit has a pseudosubstrate͞inhibitor sequence near the N terminus that binds in the active site of the C-subunit. Two cAMP-binding domains (A and B) follow the pseudosubstrate͞inhibitor sequence, and it is the A-domain of the R I ␣ isoform that contains the peripheral binding site for inhibition of the C-subunit (1, 2). Although crystal structures are available for the C-subunit in various active and inhibited forms (3), and for the cAMP-bound R I ␣(113-376) (4), no structural information is available for the holoenzyme complex consisting of the R-subunit and the C-subunit.Computational docking is a useful approach to build up the structures of larger protein-protein complexes. The program DOT computes the interaction energies of all possible (billions) translational and rotational solutions by using fast Fourier transform algorithms (5, 6). The interaction between the Csubunit and the R-subunit involves two sites, each of which contributes to the overall binding energy, and DOT did not predict a unique docking interface. To filter the results, we first tried using results from complementary mutagenesis experiments, which revealed a contact between Lys-213 in the Csubunit with Glu-143 in the R-subunit (7). This contact alone, however, was insufficient to specify a unique solution (I.T. and L.T.E., unpublished results).Amide H͞ 2 H exchange, followed by proteolytic digestion and mass spectrometry, is a powerful method to map protein-protein and protein-ligand interactions (8-11). Amide H͞ 2 H exchange c...

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“…To obtain a more complete picture of the dynamic response, other side-chain types and slower timescales need to be considered, especially since many signal transmission effects occur on slower timescales, such as with allosteric conformational changes (5,11,(56)(57)(58). Here, we show that model-free order parameters are readily obtained for aromatic groups using 13 C relaxation, and that flexibility changes from hydrogen exchange measurements provide a view of the slower changes in dynamics in eglin c. Taking all data together, there is a striking spatial co-localization of compensating changes in fast and slow dynamics, suggesting a possible link between fast fluctuations and rare opening events.…”

Section: Nih-pa Author Manuscriptmentioning

confidence: 99%

Monitoring Aromatic Picosecond to Nanosecond Dynamics in Proteins via ¹³C Relaxation: Expanding Perturbation Mapping of the Rigidifying Core Mutation, V54A, in Eglin c

Boyer

Lee

2008

Biochemistry

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Long-range effects, such as allostery, have evolved in proteins as a means of regulating function via communication between distal sites. An NMR-based perturbation mapping approach was used to more completely probe the dynamic response of the core mutation V54A in the protein eglin c by monitoring changes in ps-ns aromatic side-chain dynamics and H/D exchange stabilities. Previous side-chain dynamics studies on this mutant were limited to methyl-bearing residues, most of which were found to rigidify on the ps-ns timescale in the form of a contiguous 'network'. Here, high precision 13 C relaxation data from 13 aromatic side chains were acquired by applying canonical relaxation experiments to a newly-developed carbon labeling scheme [Teilum K. et al. (2006) J. Am. Chem. Soc. 128, 2506-2507. The fitting of model-free parameters yielded S 2 variability which is intermediate with respect to backbone and methyl-bearing side chain variability and τ e values that are approximately one nanosecond. Inclusion of the aromatic dynamic response results in an expanded network of dynamically coupled residues, with some aromatics showing increases in flexibility, which partially offsets the rigidification in methyl side chains. Using amide hydrogen exchange, dynamic propagation on a slower timescale was probed in response to the V54A perturbation. Surprisingly, regional stabilization (slowed exchange) 10-12 angstroms from the site of mutation was observed despite a global destabilization of 1.5 kcal·mol −1 . Furthermore, this unlikely pocket of stabilized residues co-localizes with increases in aromatic flexibility on the faster timescale. Because the converse is also true (destabilized residues co-localize with rigidification on the fast timescale), a plausible entropy-driven mechanism is discussed for relating co-localization of opposing dynamic trends on vastly different timescales. KeywordsNMR; dynamics; relaxation; aromatic; hydrogen exchange; eglin c; V54AMotional flexibility, or dynamics, is essential for protein stability and function (2-6). Recently, native-state protein dynamics have been thought to be crucial for mediating allosteric signaling, or stated more generally, long-range communication (7)(8)(9)(10)(11)(12)(13)(14). One of the major challenges is the experimental characterization of dynamic processes relevant to intra- † This research was supported by NIH Grant GM066009 * To whom correspondence should be addressed: University of North Carolina, School of Pharmacy, Beard Hall, CB# 7360, Chapel Hill, NC 27599-7360. drewlee@unc.edu. Phone: (919) 843-5150. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2011 March 23. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript molecular signal transduction. Towards this goal, NMR spectroscopy is uniquely suited to non-invasively characterize both structure and dynamics in a site-specific manner. Mapping flexibility by NMR is therefore an important application for understanding signaling in prote...

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Structural Characterization of Protein Kinase A as a Function of Nucleotide Binding

Cited by 51 publications

References 74 publications

Allostery in the ferredoxin protein motif does not involve a conformational switch

Allostery in the ferredoxin protein motif does not involve a conformational switch

Identification of the protein kinase A regulatory R ^I α-catalytic subunit interface by amide H/ ² H exchange and protein docking

Monitoring Aromatic Picosecond to Nanosecond Dynamics in Proteins via ¹³C Relaxation: Expanding Perturbation Mapping of the Rigidifying Core Mutation, V54A, in Eglin c

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Structural Characterization of Protein Kinase A as a Function of Nucleotide Binding

Cited by 51 publications

References 74 publications

Allostery in the ferredoxin protein motif does not involve a conformational switch

Allostery in the ferredoxin protein motif does not involve a conformational switch

Identification of the protein kinase A regulatory R I α-catalytic subunit interface by amide H/ 2 H exchange and protein docking

Monitoring Aromatic Picosecond to Nanosecond Dynamics in Proteins via 13C Relaxation: Expanding Perturbation Mapping of the Rigidifying Core Mutation, V54A, in Eglin c

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Identification of the protein kinase A regulatory R ^I α-catalytic subunit interface by amide H/ ² H exchange and protein docking

Monitoring Aromatic Picosecond to Nanosecond Dynamics in Proteins via ¹³C Relaxation: Expanding Perturbation Mapping of the Rigidifying Core Mutation, V54A, in Eglin c