Induction of Flexibility through Protein-Protein Interactions

Abstract

The dimerization/docking (D/D) domain of the cyclic AMP-dependent protein kinase (PKA) holoenzyme mediates important protein-protein interactions that direct the subcellular localization of the enzyme. Akinase anchoring proteins (AKAPs) provide the molecular scaffold for the localization of PKA. The recent solution structures of two D/D AKAP complexes revealed that the AKAP binds to a surface-exposed, hydrophobic groove on the D/D. In the present study, we present an analysis of the changes in hydrogen/deuterium exchange protection and internal motions of the backbone of the D/D when free and bound to the prototype anchoring protein, Ht31pep. We observe that formation of the complex results in significant, but small, increases in H/D exchange protection factors as well as increases in backbone flexibility, throughout the D/D, and in particular, in the hydrophobic binding groove. This unusual observation of increased backbone flexibility and marginal H/D exchange protection, despite high affinity protein-ligand interactions, may be a general effect observed for the stabilization of hydrophobic ligand/hydrophobic pocket interactions. The dimerization/docking (D/D) domain of the cyclic AMP-dependent protein kinase (PKA) holoenzyme mediates important protein-protein interactions that direct the subcellular localization of the enzyme. Akinase anchoring proteins (AKAPs) provide the molecular scaffold for the localization of PKA. The recent solution structures of two D/D AKAP complexes revealed that the AKAP binds to a surface-exposed, hydrophobic groove on the D/D. In the present study, we present an analysis of the changes in hydrogen/deuterium exchange protection and internal motions of the backbone of the D/D when free and bound to the prototype anchoring protein, Ht31pep. We observe that formation of the complex results in significant, but small, increases in H/D exchange protection factors as well as increases in backbone flexibility, throughout the D/D, and in particular, in the hydrophobic binding groove. This unusual observation of increased backbone flexibility and marginal H/D exchange protection, despite high affinity protein-ligand interactions, may be a general effect observed for the stabilization of hydrophobic ligand/hydrophobic pocket interactions. cyclic AMP-dependent protein kinase A kinase anchoring protein dimerization/docking Protein-protein interactions control many critical functions in biology, ranging from tight binding antibody-antigen recognition events to transient interactions between enzymes in a signaling pathway. These interactions can be complex; there are sometimes a number of diverse proteins that can interact with a particular target molecule (1Wells J.A. de Vos A.M. Annu. Rev. Biochem. 1996; 65: 609-634Crossref PubMed Scopus (255) Google Scholar, 2Pawson T. Scott J.D. Science. 1997; 278: 2075-2080Crossref PubMed Scopus (1900) Google Scholar). Elucidation of key intermolecular contacts between protein partners can aid in the development of small molecule inhibitors and/or promoters of these important interactions, which in turn control function. A particularly interesting area in biology today is the investigation of the molecular mechanisms of the assembly/disassembly of signaling networks in response to a specific cellular signal (3Hunter T. Cell. 2000; 100: 113-127Abstract Full Text Full Text PDF PubMed Scopus (2269) Google Scholar). Indeed, the spatio-temporal compartmentalization of signaling molecules affords biological control by poising interacting partners in close proximity to substrate(s) and/or regulatory elements (4Lester L.B. Scott J.D. Recent Prog. Horm. Res. 1997; 52: 409-429PubMed Google Scholar). Targeting of the cyclic AMP-dependent protein kinase (PKA)1 holoenzyme through interactions with Akinaseanchoring proteins (AKAPs) has emerged as an important modulator of PKA activity in diverse tissues (5Colledge M. Scott J.D. Trends Cell Biol. 1999; 9: 216-221Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar). The PKA holoenzyme consists of a regulatory subunit (R2) dimer and two catalytic (C) subunits (6Taylor S.S. Buechler J.A. Yonemoto W. Annu. Rev. Biochem. 1990; 59: 971-1005Crossref PubMed Scopus (958) Google Scholar). Phosphorylation of target proteins is carried out by the C subunit, whereas the N-terminal 45 residues of the R subunit mediates both dimerization and subcellular localization via AKAP recognition (7Hausken Z.E. Coghlan V.M. Hastings C.A. Reimann E.M. Scott J.D. J. Biol. Chem. 1994; 269: 24245-24251Abstract Full Text PDF PubMed Google Scholar, 8Hausken Z.E. Dell'Acqua M.L. Coghlan V.M. Scott J.D. J. Biol. Chem. 1996; 271: 29016-29022Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Hence, the N-terminal functional domain is termed aD/D motif because it dimerizes anddocks to anchoring partners. Solution structural studies revealed that the type IIα D/D of PKA packs into an antiparallel, dimeric X-type four-helix bundle, with a surface-exposed hydrophobic groove that is the site of anchoring interactions (9Newlon M.G. Roy M. Morikis D. Hausken Z.E. Coghlan V. Scott J.D. Jennings P.A. Nat. Struct. Biol. 1999; 6: 222-227Crossref PubMed Scopus (184) Google Scholar, 10Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Crossref PubMed Scopus (181) Google Scholar). PKA interacts with a diverse family of proteins. Sequence alignment of the identified AKAPs, to date, reveals no specific recognition sequence for the D/D. However, a conserved structure consistent with an amphipathic helix was predicted, and has been demonstrated in recent solution structural studies of a peptide derivative of the prototypic AKAP human thyroid anchoring protein Ht31 (residues 493–515 and designated Ht31pep) (7Hausken Z.E. Coghlan V.M. Hastings C.A. Reimann E.M. Scott J.D. J. Biol. Chem. 1994; 269: 24245-24251Abstract Full Text PDF PubMed Google Scholar, 11Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar, 12Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar). This peptide derivative of Ht31 exhibits a nanomolar binding affinity for the type II D/D (12Carr D.W. Stofko-Hahn R.E. Fraser I.D. Cone R.D. Scott J.D. J. Biol. Chem. 1992; 267: 16816-16823Abstract Full Text PDF PubMed Google Scholar, 13Vijayaraghavan S. Liberty G.A. Mohan J. Winfrey V.P. Olson G.E. Carr D.W. Mol. Endocrinol. 1999; 13: 705-717Crossref PubMed Scopus (0) Google Scholar) via hydrophobic-hydrophobic interactions between the surface-accessible hydrophobic groove on the D/D and the hydrophobic face of the AKAP-derived amphipathic helix (10Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Crossref PubMed Scopus (181) Google Scholar, 14Carr D.W. Stofko-Hahn R.E. Fraser I.D. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D. J. Biol. Chem. 1991; 266: 14188-14192Abstract Full Text PDF PubMed Google Scholar). In an effort to gain a better understanding of the physicochemical basis for PKA-AKAP interactions, we initiated hydrogen/deuterium (H/D) exchange and backbone relaxation studies of the D/D free and in complex with Ht31pep. In contrast to recent work described by Powell et al. (15Powell K.D. Ghaemmaghami S. Wang M.Z. Ma L. Oas T.G. Fitzgerald M.C. J. Am. Chem. Soc. 2002; 124: 10256-10257Crossref PubMed Scopus (127) Google Scholar) using H/D exchange to measure ligand-binding affinities, we observe only modest changes in the H/D protection factors upon complex formation, despite the nanomolar binding affinity of Ht31 for the D/D (11Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar). Unexpectedly, we also find that backbone flexibility in the binding interface of the D/Dincreases in the Ht31pep complex. We propose that the increase in backbone mobility and display of modest changes in H/D exchange protection factors upon high affinity ligand binding may be a general effect observed for proteins that use solvent accessible hydrophobic surfaces to recognize diverse binding partners. The D/D·Ht31peppeptide complex was prepared as described previously (16Newlon M.G. Roy M. Hausken Z.E. Scott J.D. Jennings P.A. J. Biol. Chem. 1997; 272: 23637-23644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The Ht31pep peptide was obtained from PeptidoGenic Research and Co. (Livermore, CA). The stoichiometry of binding for classical AKAPs is one AKAP per R subunit dimer (11Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar). The apo-D/D was prepared as a 0.25 mm (0.5 mm monomer) sample in 20 mm sodium phosphate buffer, 90% H2O, 10% D2O, pH 4.0. Relaxation experiments were collected on either a 0.25 mm (0.50 mm monomer apo) or a 0.50 mm (1.0 mm monomer complex) sample. Samples for hydrogen/deuterium exchange studies were collected on 0.25–1.0 mm protein solutions. Hydrogen/deuterium exchange experiments were initiated by introducing the protein samples into deuterated buffer via a QuikChange gel chromatography step (Roche Molecular Biochemicals). A series of two-dimensional 1H-15N heteronuclear single quantum coherence spectra (17Mori S. Abeygunawardana C. Johnson M.O. van Zijl P.C. J. Magn. Reson. B. 1995; 108: 94-98Crossref PubMed Scopus (569) Google Scholar) were collected on a Bruker DMX500 spectrometer at 0, 16, 30, 60, 100, 300, 1080, 1560, and 2880 min after the introduction of the sample into D2O buffer. Calibration of the individual spectra to correct for protein concentration differences between samples was achieved by normalizing the data to the intensity of the non-exchanging aliphatic resonance of Val20 Hγ 2* in a one-dimensional 1H spectrum taken directly after the completion of the heteronuclear single quantum coherence experiments. The time-dependent change in the cross-peak intensity (volume) of each amide proton resonance was found to be exponential. Fitting of each of the observed decay curves to an exponential decay function allowed the extraction of the residue specific experimental exchange rate,kobs, according to, I=e−kobs×tEquation 1 where I is the observed cross-peak intensity (volume) at time t. Fitting of data, whether normalized or non-normalized, volume or intensity, from 600 or 500 MHz spectrometer gave identical rates (data not shown). Residue-specific protection factors,P, for individual amide protons were from the where is the protection the for sequence L. PubMed Scopus Google and the observed for the solvent exchange of the amide A general of the exchange can be described as Chem. PubMed Scopus Google where and and deuterated backbone C the and the and are The for the exchange for a specific amide proton upon the and which exchange L. PubMed Scopus Google Scholar, R.G. PubMed Scopus Google Scholar). the for the is by the the exchange step is the for we the the is the exchange to where the for the This is as the these and a well an free of can be from the protection factors according to the where R is the the in and is the protection R.G. PubMed Scopus Google Scholar). Relaxation experiments were collected at on Bruker DMX500 and using a The and were with that use for coherence with R. S.M. T. J.D. 1994; PubMed Scopus Google Scholar, J. Magn. Reson. 1992; Scholar, J. Chem. Scopus Google Scholar). The and relaxation experiments were collected as a time series of two 1H-15N with 300, 100, and for and and for where to the in the were using the and the for the amide 1H-15N were with M. Scholar). and relaxation rates were by the time series to a single exponential decay function. The in the rates are as the in the were from the of the of the with and proton were both from experiments and from of the of the in the spectra R. S.M. T. J.D. 1994; PubMed Scopus Google Scholar). Relaxation of an amide is by with the and of the of the are by the which is to the relaxation and of Scholar). The J. Am. Chem. Soc. Scopus Google Scholar, P.C. A.M. 1990; PubMed Scopus Google Scholar) the of the and time of the motions by the in of the the of internal motions of each time for internal and time of the an J. Magn. Reson. B. 1994; PubMed Scopus Google Scholar, J. Am. Chem. Soc. 1995; Scopus Google the can be as a function of the between the and the of the of the We data at 500 and 600 MHz for of a internal motions for each residue P.C. A.M. 1990; PubMed Scopus Google Scholar). the was the molecular and the internal were of the function A.M. M. J. Mol. Biol. 1995; PubMed Scopus Google Scholar). The free were using the and by were for the D/D in the Ht31pep complex for residues and where it was not to (10Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Crossref PubMed Scopus (181) Google Scholar). In the analysis was by to (residues and differences were observed in the for residues and a of residues in the D/D and in the and and residues in the Ht31 complex were from analysis because of signal intensity and/or resonance the peptide Ht31pep binds to in a one peptide to one dimer it into the complex in changes in the D/D dimer upon peptide binding (10Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Crossref PubMed Scopus (181) Google Scholar, M.G. Roy M. Hausken Z.E. Scott J.D. Jennings P.A. J. Biol. Chem. 1997; 272: 23637-23644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). on both the and of and in exchange upon peptide binding The changes in amide exchange upon AKAP binding in helix through which the hydrophobic that is the site of direct AKAP studies that residues and important for Ht31pep binding (11Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google but these residues are in both the and the complex. the of the molecule and are from the AKAP binding increases in exchange protection factors are also observed in upon complex formation exchange in a differences in observed between the free and the of The data between the to changes that are not the for A and are by and residues are by a in the differences in the protection factors and accessible area observed between the free and the of residues are by a in the The accessible area was with R. M. J. Mol. 1996; Scopus Google 1H-15N resonance of the and D/D were as described previously (16Newlon M.G. Roy M. Hausken Z.E. Scott J.D. Jennings P.A. J. Biol. Chem. 1997; 272: 23637-23644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Relaxation and were obtained from the analysis of proton spectra of the free and the complex. were collected at two 500 and 600 of amide proton resonance were of for the of the cross-peak in the individual The relaxation are in We observe a in the upon complex formation for of the residues in the hydrophobic binding and a increase in the for the important residues and S. Liberty G.A. Mohan J. Winfrey V.P. Olson G.E. Carr D.W. Mol. Endocrinol. 1999; 13: 705-717Crossref PubMed Scopus (0) Google Scholar, 14Carr D.W. Stofko-Hahn R.E. Fraser I.D. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D. J. Biol. Chem. 1991; 266: 14188-14192Abstract Full Text PDF PubMed Google These upon complex formation to a where the domain are the for the free and Ht31pep as at 500 and 600 MHz II and of the relaxation rates for the and Ht31pep complex a to the The for is that observed for the when at 500 but is as at 600 The relaxation rates for and the complex 500 and 600 are experimental but individual residue rates changes upon complex formation, the of of the relaxation for the free and in a We a analysis J. Am. Chem. Soc. Scopus Google Scholar) of the data in an effort to relaxation in of is observed in the complex and were where (16Newlon M.G. Roy M. Hausken Z.E. Scott J.D. Jennings P.A. J. Biol. Chem. 1997; 272: 23637-23644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The observed relaxation an A we a of residues in analysis of the complex. The experimental data and 600 were either an or an molecular The of were from the one of the PubMed Scopus Google using the by and the of the free or (16Newlon M.G. Roy M. Hausken Z.E. Scott J.D. Jennings P.A. J. Biol. Chem. 1997; 272: 23637-23644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The of were and the the and was for The were as the and the the of and for the free and A of the data at 500 and 600 MHz allowed the of a for of the that not be by a are in the and/or of the The free are in and a these is for the free and in a The in the type IIα D/D of PKA to an X-type four-helix with an of and interactions the (9Newlon M.G. Roy M. Morikis D. Hausken Z.E. Coghlan V. Scott J.D. Jennings P.A. Nat. Struct. Biol. 1999; 6: 222-227Crossref PubMed Scopus (184) Google Scholar, 10Newlon M.G. Roy M. Morikis D. Carr D.W. Westphal R. Scott J.D. Jennings P.A. EMBO J. 2001; 20: 1651-1662Crossref PubMed Scopus (181) Google Scholar, J. Mol. Biol. 1994; PubMed Scopus Google Scholar). The protein is by hydrophobic interactions between that the dimer In the D/D a hydrophobic groove the solvent of the interface of D.W. Stofko-Hahn R.E. Fraser I.D. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D. J. Biol. Chem. 1991; 266: 14188-14192Abstract Full Text PDF PubMed Google Scholar). The hydrophobic of groove each and are well in the solution despite solvent This is an but of the D/D, which in protein-protein interactions with a diverse family of anchoring proteins J.D. S. Mol. Endocrinol. 1994; PubMed Scopus Google Scholar). studies on a family of anchoring proteins a sequence of 20 that a high of amphipathic helix formation that is the site for D/D binding R. Jennings P.A. Scott J.D. Google Scholar). Ht31pep has emerged as the prototypic AKAP and structural studies the of hydrophobic from the amphipathic helix of Ht31 for D/D anchoring interactions (11Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar). Ht31pep is a for the of PKA anchoring (11Carr D.W. Hausken Z.E. Fraser I.D. Stofko-Hahn R.E. Scott J.D. J. Biol. Chem. 1992; 267: 13376-13382Abstract Full Text PDF PubMed Google Scholar). Ht31pep a of AKAP partners in it as the for understanding AKAP recognition by PKA D.W. Stofko-Hahn R.E. Fraser I.D. Bishop S.M. Acott T.S. Brennan R.G. Scott J.D. J. Biol. Chem. 1991; 266: 14188-14192Abstract Full Text PDF PubMed Google Scholar, J.D. S. Mol. Endocrinol. 1994; PubMed Scopus Google Scholar). In the changes in the protection factors of the protons upon ligand binding can be to binding that the protein is amide exchange in the (15Powell K.D. Ghaemmaghami S. Wang M.Z. Ma L. Oas T.G. Fitzgerald M.C. J. Am. Chem. Soc. 2002; 124: 10256-10257Crossref PubMed Scopus (127) Google Scholar, S. Fitzgerald M.C. Oas T.G. 2000; PubMed Scopus Google Scholar). is the exchange for backbone amide protons in proteins where the is and the exchange is A pH to for the of exchange in the D/D was not the that the protein to pH from (16Newlon M.G. Roy M. Hausken Z.E. Scott J.D. Jennings P.A. J. Biol. Chem. 1997; 272: 23637-23644Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). However, analysis of the exchange upon peptide binding no for we results an the observation of Powell et al. (15Powell K.D. Ghaemmaghami S. Wang M.Z. Ma L. Oas T.G. Fitzgerald M.C. J. Am. Chem. Soc. 2002; 124: 10256-10257Crossref PubMed Scopus (127) Google Scholar) where the observed changes in amide proton exchange rates are in with from the ligand binding affinities, we found that the of the binding from the observed changes in protection factors the Ht31pep binding affinity I and data that the D/D either through motions J. A.M. M. PubMed Scopus (60) Google Scholar) or as a of which in exchange J. Mol. Biol. 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Scopus Google Scholar) in which the internal motions are described by the and the internal that are in the structures the residues in the and the of the C were better described with and for and internal and of an and to for motions was for a residues in the helix and for the free D/D and and for the and for one in the helix in both This the hydrophobic binding and may exchange consistent with the of protein D. Oas T.G. S. PubMed Scopus Google Scholar). A effect was also observed in studies on the domain of I bound to a L. C. 1996; PubMed Scopus Google Scholar). Protein-protein interactions control a diverse of biological we not a understanding of target the molecular basis for protein target binding is by a of factors binding as well as changes in and backbone of the interacting partners J.D. Nat. Struct. Biol. 1999; 6: PubMed Scopus Google Scholar, J. M. Nat. Struct. 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Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). structural studies of anchoring the of structural of in anchoring interactions. of an important recognition for PKA through D/D to diverse anchoring partners. We and Roy for critical of the for Morikis for to the exchange and the for