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• W2066094617 abstract "The solution structure of an extended pleckstrin homology (PH) domain from the β-adrenergic receptor kinase is obtained by high resolution NMR. The structure establishes that the β-adrenergic receptor kinase extended PH domain has the same fold and topology as other PH domains, and there are several unique features, most notably an extended C-terminal α-helix that behaves as a molten helix, and a surface charge polarity that is extensively modified by positive residues in the extended α-helix and the C terminus. These observations complement biochemical evidence that the C-terminal portion of this PH domain participates in protein-protein interactions with Gβγ subunits. This suggests that the C-terminal segment of the PH domain may function to mediate protein-protein interactions with the targets of PH domains. The solution structure of an extended pleckstrin homology (PH) domain from the β-adrenergic receptor kinase is obtained by high resolution NMR. The structure establishes that the β-adrenergic receptor kinase extended PH domain has the same fold and topology as other PH domains, and there are several unique features, most notably an extended C-terminal α-helix that behaves as a molten helix, and a surface charge polarity that is extensively modified by positive residues in the extended α-helix and the C terminus. These observations complement biochemical evidence that the C-terminal portion of this PH domain participates in protein-protein interactions with Gβγ subunits. This suggests that the C-terminal segment of the PH domain may function to mediate protein-protein interactions with the targets of PH domains. G protein-coupled receptor kinases (GRKs) 1The abbreviations used are: GRK, G protein-coupled receptor kinase; PH, pleckstrin homology; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; Ins(1,4,5)P3, myo-inositol 1,4,5-trisphosphate; PTB, phosphotyrosine binding domain; βARK, β-adrenergic receptor kinase; GST, glutathione S-transferase; r.m.s.d., root mean square deviation; NOE, nuclear Overhauser effect; PLC, phospholipase C; HCCH, 1H-13C-13C-1H multidimensional correlation spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser spectroscopy; ROESY, rotating frame NOESY; HMQC, heteronuclear multiquantum coherence spectroscopy; HTQC, heteronuclear triple quantum coherence spectroscopy; HSQC, heteronuclear single quantum coherence spectroscopy; HOHAHA, homonuclear Hartmann-Hahn (correlation) spectroscopy (The acronyms HNCA, HNCO, CBCANH, CBCA(CO)NH, and WATERGATE refer to pulse sequence selection programs and are referred to in the text. DIANA, DYANA, REDAC, and ECEPP are computer analysis programs.) 1The abbreviations used are: GRK, G protein-coupled receptor kinase; PH, pleckstrin homology; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; Ins(1,4,5)P3, myo-inositol 1,4,5-trisphosphate; PTB, phosphotyrosine binding domain; βARK, β-adrenergic receptor kinase; GST, glutathione S-transferase; r.m.s.d., root mean square deviation; NOE, nuclear Overhauser effect; PLC, phospholipase C; HCCH, 1H-13C-13C-1H multidimensional correlation spectroscopy; TOCSY, total correlation spectroscopy; NOESY, nuclear Overhauser spectroscopy; ROESY, rotating frame NOESY; HMQC, heteronuclear multiquantum coherence spectroscopy; HTQC, heteronuclear triple quantum coherence spectroscopy; HSQC, heteronuclear single quantum coherence spectroscopy; HOHAHA, homonuclear Hartmann-Hahn (correlation) spectroscopy (The acronyms HNCA, HNCO, CBCANH, CBCA(CO)NH, and WATERGATE refer to pulse sequence selection programs and are referred to in the text. DIANA, DYANA, REDAC, and ECEPP are computer analysis programs.) are a unique family of serine-threonine kinases, which are responsible for activator-dependent phosphorylation of G protein receptors and provide rapid desensitization of the agonist occupied receptors (1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (468) Google Scholar). The GRKs are recognized to have three functional components: an N-terminal section believed to interact directly with the seven-trans-membrane helical receptor protein and/or other membrane targets, a central section, which is the catalytic domain, and a C-terminal section containing a generally conserved autophosphorylation region and a variable region that mediates membrane association by various means. In GRK2 (also known as β-adrenergic receptor kinase-1) or GRK3 (β-adrenergic receptor kinase-2), the C-terminal variable region contains a pleckstrin homology (PH) domain (2Mayer B.J. Ren R. Clark K.L. Baltimore D. Cell. 1993; 73: 629-630Abstract Full Text PDF PubMed Scopus (378) Google Scholar, 3Haslam R.J. Koide H.B. Hemmings B.A. Nature. 1993; 363: 309-310Crossref PubMed Scopus (382) Google Scholar), conferring binding specificity to Gβγ proteins (reviewed in Ref.1Premont R.T. Inglese J. Lefkowitz R.J. FASEB J. 1995; 9: 175-182Crossref PubMed Scopus (468) Google Scholar). The PH domain family (reviewed in Refs. 4Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2211) Google Scholar, 5Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (922) Google Scholar, 6Shaw G. BioEssays. 1996; 18: 35-46Crossref PubMed Scopus (252) Google Scholar) appears to be a very large family of structurally homologous protein domains of moderate to low sequence similarity. The PH domain is believed to play a role in intracellular signal transduction, and the functional role of the PH domain has been characterized for several systems. In phospholipase Cδ, the PH domain has a high affinity (Kd < 1 μm) site for phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) (7Lemmon M.A. Ferguson K.M. O'Brien R. Sigler P.B. Schlessinger J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10472-10476Crossref PubMed Scopus (470) Google Scholar), which forms a crystallographically observed, well defined structural interaction (8Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (522) Google Scholar). Other PH domains have different lipid specificities, and a well defined set of binding motifs does not readily emerge (9Cowburn D. Kuriyan J. Heldin C.-H. Purton M. Signal Transduction. Chapman & Hall, London1996: 127-142Google Scholar, 10Harlan J.E. Yoon H.S. Hajduk P.J. Fesik S.W. Biochemistry. 1995; 34: 9859-9864Crossref PubMed Scopus (105) Google Scholar, 11Hyvonen M. Macias M.J. Nilges M. Oschkinat H. Saraste M. Wilmanns M. EMBO J. 1995; 14: 4676-4685Crossref PubMed Scopus (303) Google Scholar, 12Zheng J. Cahill S.M. Lemmon M.A. Fushman D. Schlessinger J. Cowburn D. J. Mol. Biol. 1996; 255: 14-21Crossref PubMed Scopus (216) Google Scholar, 13Franke T.F. Kaplan D.R. Cantley L.C. Toker A. Science. 1997; 275: 665-668Crossref PubMed Scopus (1290) Google Scholar, 14Klippel A. Kavanaugh W.M. Pot D. Williams L.T. Mol. Cell. Biol. 1997; 17: 338-344Crossref PubMed Scopus (443) Google Scholar, 15Salim K. Bottomley M.J. Querfurth E. Zvelebil M.J. Gout I. Scaife R. Margolis R.L. Gigg R. Smith C.I. Driscoll P.C. Waterfield M.D. Panayotou G. EMBO J. 1996; 15: 6241-6250Crossref PubMed Scopus (487) Google Scholar). One hypothesis is that PH domains present a framework with a polymorphic surface used for specific recognition, analogous to immunoglobulins (5Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (922) Google Scholar, 9Cowburn D. Kuriyan J. Heldin C.-H. Purton M. Signal Transduction. Chapman & Hall, London1996: 127-142Google Scholar). In addition, the overall fold of the PH domain was observed to be common with that of the PTB (phosphotyrosine binding) domain (16Zhou M.M. Fesik S.W. Prog. Biophys. Mol. Biol. 1995; 64: 221-235Crossref PubMed Scopus (20) Google Scholar, 17Eck M.J. Dhe-Paganon S. Trub T. Nolte R.T. Shoelson S.E. Cell. 1996; 85: 695-705Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar), a protein domain that shows little sequence homology to PH domains. In light of these developments, it is of significance to establish whether the nominal PH domain of GRK-2 (β-adrenergic receptor kinase (βARK-1)), which clearly binds (both in vivo and in vitro) to a protein partner, Gβγ subunits of the heterotrimeric G protein family (18Touhara K. Inglese J. Pitcher J.A. Shaw G. Lefkowitz R.J. J. Biol. Chem. 1994; 269: 10217-10220Abstract Full Text PDF PubMed Google Scholar), truly has the common structural motif of the PH/PTB domains, and what the relationship of putative lipid and protein binding sites might be in such a structure. In this paper, we present the solution structure of an extended PH domain from human βARK1, determined at 0.4 Å r.m.s.d. by high resolution NMR using heteronuclear triple resonance methods. Although the overall fold and topology clearly establishes that the βARK1 extended PH domain is of the same family as other PH domains, there are several significant alterations (most notably an extension of the C-terminal α-helix, which in solution presents as a “molten helix” having a clear gradient of mobility) on the subnanosecond, as well as millisecond to microsecond, time scales, increasing toward the free C terminus. The polarity of the surface charge observed in other PH domains is altered by positively charged residues in the extended α-helix. This unusual clustering may be complemented by a highly negatively charged area on Gβγ subunits. Although a direct study of the Gβγ/PH domain complex is beyond the range of current NMR technology, the structure presented here supports a model in which the C-terminal portion of βARK PH domain in particular, and PH domains in general, participate in protein-protein interactions. Recombinant human βARK PH domain (hβARK1-(556–670)) was obtained by GST fusion expression from pGEX-2T (Pharmacia Biotech Inc.) in BL21 (DE3) Escherichia coli cells (Novagen, Madison, WI) and subsequent bacterial expression and protein purification as described previously (19Mahadevan D. Thanki N. Singh J. McPhie P. Zangrilli D. Wang L.M. Guerrero C. LeVine III, H. Humblet C. Saldanha J. Gutkind J.S. Najmabadi-Haske T. Biochemistry. 1995; 34: 9111-9117Crossref PubMed Scopus (62) Google Scholar) on a larger scale. The full-length hβARK1 cDNA clone was provided by Dr. Antonio DeBlasi (Mario Negri Sud, Santa Maria Imbaro, Italy). The sequence of the 119-residue construct used in the present study is shown in Fig. 1. It contains both the PH domain and the Gβγ-binding motif (20Touhara K. Koch W.J. Hawes B.E. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 17000-17005Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). The first four residues are not from the natural sequence. Uniform 15N and15N,13C labeling was achieved by growing the cells in M9 minimal medium using standard procedures. Solutions used for NMR studies contained 1–2 mm protein in 10 mm acetate buffer at pH 4.5 (uncorrected for isotope effects), 0.02% sodium azide, 1 mm[U-2H]EDTA, 5 mm[U-2H]dithiothreitol, and 10%2H2O in the H2O samples. These low salt and low pH conditions were necessary to prevent protein aggregation. CD data indicated no changes in the protein secondary structure, between the buffer used for NMR studies and phosphate-buffered saline, pH 7.2. The external 1H chemical shift reference used was sodium 2,2-dimethyl-2-silapentane-5-sulfonate, and indirect referencing was used for 15N (21Live D.H. Davis D.G. Agosta W.C. Cowburn D. J. Am. Chem. Soc. 1984; 106: 1939-1941Crossref Scopus (234) Google Scholar) and13C. Spectra were essentially identical among several preparations of the PH domain. NMR experiments were run on Bruker DMX-500 and DMX-600 spectrometers. Quadrature detection was achieved by the States or States-time proportional phase incrementation methods. Some of the pulse schemes implemented pulse field gradients for coherence selection (HCCH-TOCSY, 13C-separated NOESY-HMQC), and some used the sensitivity enhancement method (HSQC, heteronuclear NOE) (22Palmer A.G. Cavanaugh J. Wright P.E. Rance M. J. Magn. Reson. 1991; 93: 151-170Google Scholar). The water signal was suppressed either by the WATERGATE method (23Sklenar V. Piotto M. Leppik R. Sandek V. J. Magn. Reson. 1993; 102a: 241-245Crossref Scopus (1105) Google Scholar) or by using selective on-resonance irradiation during a relaxation delay of ∼1.3 s. Experiments were run at 35 °C with sweep widths of 8000 and 2000 Hz for 1H and 15N (at 600 MHz), respectively, unless indicated otherwise. The homonuclear experiments, HOHAHA and NOESY, were run in both H2O and 2H2O using standard pulse sequences and phase cycling. A range of t1increments from 200 to 512, each consisting of 2048 complex points, was typically acquired with 32–128 scans/increment. The heteronuclear experiments consisted of two-dimensional HSQC (1H-15N and 1H-13C), HSQC-J, and HTQC, three-dimensional CBCA(CO)NH, CBCANH, HNCA, HN(CO)CA, HNCO, HCCH-TOCSY, and 13C-separated NOESY-HMQC, and two- and three-dimensional 15N-separated NOESY-HMQC (in H2O and in 2H2O) and HOHAHA-HMQC. The mixing time in the NOESY-HMQC experiments was 100 and 150 ms, and the spin lock duration was 30 ms in the HOHAHA-HMQC and 19 ms and 6 ms in the HCCH-TOCSY. The three-dimensional spectra were recorded with a 32 × 100 × 1000 hypercomplex matrix, with 32 scans/increment. The degree of amide hydrogen protection was assessed (a) by measuring hydrogen-deuterium exchange rates by following the intensity of cross-peaks in HMQC experiments after exchanging a fully protonated, lyophilized sample with 99.996%2H2O, and (b) by comparison of cross-peak intensities in two HSQC experiments, with and without water presaturation (24Morelli M.A.C. Stier G. Gibson T. Joseph C. Musco G. Pastore A. Trave G. FEBS Lett. 1995; 358: 193-198Crossref PubMed Scopus (33) Google Scholar). The three-bond H′-Hα coupling was assessed by the method of (25Kay L.E. Bax A. J. Magn. Reson. 1990; 86: 110-126Google Scholar). Heteronuclear 15N{1H} NOEs were measured using standard methods as described elsewhere (26Fushman D. Cahill S. Cowburn D. J. Mol. Biol. 1997; 266: 173-194Crossref PubMed Scopus (202) Google Scholar). Two-dimensional H2O-selective heteronuclear15N-edited ROESY experiments (27Dalvit C. Hommel U. J. Biomol. NMR. 1995; 5: 306-310Crossref PubMed Scopus (30) Google Scholar) were performed to map those amide hydrogens in the βARK PH domain that are exposed to and interacting with water molecules. Signal processing and assignment were done as discussed previously (26Fushman D. Cahill S. Cowburn D. J. Mol. Biol. 1997; 266: 173-194Crossref PubMed Scopus (202) Google Scholar, 28Fushman D. Cahill S. Lemmon M.A. Schlessinger J. Cowburn D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 816-820Crossref PubMed Scopus (0) Google Scholar). Structure calculations used DIANA with REDAC strategy (29Guntert P. Wuthrich K. J. Biomol. NMR. 1991; 1: 447-456Crossref PubMed Scopus (335) Google Scholar) or DYANA (30Guentert P. Mumenthaler C. Wuthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2537) Google Scholar) with ECEPP stereochemistry, with structurally significant constraints of 1956 upper and 76 lower distance bounds (from ∼3000 NOEs), 38 hydrogen bonds chosen within strands or helix with slowed exchange, 99 φ-angle constraints derived from 3 JHNHα coupling constants, and 99 ψ-angle constraints (derived from Cα chemical shift data) corresponding to conservative ranges of allowed torsion angles, in those regions of strand or helix that were well defined (Fig. 2). All peptide bonds were assumed to be trans. A final selection of 20 structures from 400 starting structures was done by using the lowest target functions (the ensemble statistics are shown in Table I). DIANA and DYANA use no assumptions about protein energetics, other than van der Waals repulsion; structures are unrefined and only adjusted by rotation/translation for comparison purposes. Structures were aligned using XPLOR or in-house software written in MATLAB (MathWorks) and displayed and analyzed with the INSIGHTII package (Biosym) or with MOLMOL (31Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14: 51-55Crossref PubMed Scopus (6454) Google Scholar).Table IRoot-mean-square deviation from the mean structure calculated for the ensemble of 20 structures of the βARK1 PH domainSelected residuesSelected atomsr.m.s.d.ÅAll 119 residuesBackbone heavy atoms3.80 ± 0.65Residues 560–658, floppy tails clipped offBackbone heavy atoms1.08 ± 0.40Residues 560–658, floppy tails clipped offAll heavy atoms1.38 ± 0.41All elements of secondary structure1-aStrands β1 through β7 and the α-helix. There were no NOE violations of upper limits >1 Å, and eight in the ensemble of 20 structures >0.5 Å, and eight dihedral angle violations >5 °.Backbone heavy atoms0.38 ± 0.09All elements of secondary structureAll heavy atoms0.82 ± 0.16All elements of secondary structureAll atoms1.20 ± 0.17All β-strandsBackbone heavy atoms0.44 ± 0.11α-HelixBackbone heavy atoms0.31 ± 0.101-a Strands β1 through β7 and the α-helix. There were no NOE violations of upper limits >1 Å, and eight in the ensemble of 20 structures >0.5 Å, and eight dihedral angle violations >5 °. Open table in a new tab Pairwise comparison of the PH/PTB domain structures was performed by superposition of the backbone heavy atoms (N, Cα, C′, and O) of the residues from regions of regular secondary structure, as indicated by boxes in Table II. The α-helical insertions in the loop regions of the PH domains were not taken into account. The alignment was done by direct calculation of r.m.s.d. values and optimized by relative shift of the protein sequences within each secondary structure element (β1–β7 strands, α-helix), as well as by adding or removing individual residues. The resulting alignment and r.m.s.d. values are presented in Tables IIand III, respectively.Table IIStructure-based alignment of the PH/PTB domains Open table in a new tab Table IIIPairwise root-mean-square deviations (in Å) between the PH/PTB domain structuresView Large Image Figure ViewerDownload (PPT)3-a Dyn (A) and (B) refer to the crystal structures of the two monomers in the dynamin PH domain dimer observed in the crystallographic studies (35Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (240) Google Scholar). Open table in a new tab 3-a Dyn (A) and (B) refer to the crystal structures of the two monomers in the dynamin PH domain dimer observed in the crystallographic studies (35Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (240) Google Scholar). The backbone dynamics were assessed via 15N spin relaxation studies comprisingT1, T2, and heteronuclear steady state NOE measurements using previously described protocols (26Fushman D. Cahill S. Cowburn D. J. Mol. Biol. 1997; 266: 173-194Crossref PubMed Scopus (202) Google Scholar). Fifteen two-dimensional spectra with the relaxation delays of 4 (×2), 200, 400 (×2), 600, 900 (×2), and 1200 ms (positive initial15N magnetization), and 4, 200 (×2), 400, 600 (×2), and 900 ms (negative initial 15N magnetization) were acquired in the alternate-sign T1 experiment (duplicate experiments are indicated by ×2) (26Fushman D. Cahill S. Cowburn D. J. Mol. Biol. 1997; 266: 173-194Crossref PubMed Scopus (202) Google Scholar). Eleven two-dimensional spectra were collected for the T2 measurements, with the relaxation delays of 8 (×2), 16, 32 (×2), 48, 64 (×2), 80, 96 (×2), 112, 128 (×2), and 160 ms. The heteronuclear {1H}15N steady state NOEs were assessed as a ratio of cross-peak intensities in the experiments with and without proton presaturation. The relaxation data analysis was performed using programs RELAXFIT and DYNAMICS (26Fushman D. Cahill S. Cowburn D. J. Mol. Biol. 1997; 266: 173-194Crossref PubMed Scopus (202) Google Scholar), extended to include anisotropic character of the overall motion of the protein (32Tjandra N. Szabo A. Bax A. J. Am. Chem. Soc. 1996; 118: 6986-6991Crossref Scopus (316) Google Scholar). hβARK-PH domain corresponding to residues 556–670 of human βARK1 (Fig. 1) was produced in E. coli and isolated as a GST fusion protein, cleaved, and purified. Solubility and stability limited observation to a narrow range of conditions, and the majority of studies were conducted in 20 mm acetate buffer at pH 4.5, 35 °C. Under these conditions, binding of the construct to Gβγ is maintained (data not shown). The 546–670 construct was also produced, and NMR spectra indicated that the additional N-terminal residues did not belong to the domain fold, and were apparently unstructured. It was concluded that the first construct contained the essential domain. Assignment used standard triple resonance methods, complemented by study of the [U 13C, 15N; 12C,14N-Met]PH domain to help identify methionine residues that underwent partial oxidation during sample preparation. Assignment and NOE data are summarized in Fig. 2. In Figs. 3 and4, the overall fold and the electrostatic potential of the hβARK PH domain are shown, along with the PH domain of PLCδ (8Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (522) Google Scholar) for comparison. The topology of the fold is typical for PH domains, and consists of seven β-strands forming a β-sandwich flanked on one end by a C-terminal α-helix. The termini of the construct are disordered and highly flexible, with large amplitudes of backbone motion on a nanosecond time scale (Fig. 3). The β1/β2 and β3/β4 loops are disordered and display increased amplitudes of backbone dynamics on a subnanosecond to nanosecond time scale, as well as motions on a millisecond to microsecond time range (data not shown).Figure 4The effect of the C terminus on the electrostatic potential of the hβARK1 PH domain, and comparison with the PLCδ PH domain. Surfaces are contoured at −2kT/e (red) and 2kT/e (blue) (GRASP; Ref. 56Nicholls A. Sharp K.A. Honig B. Proteins. 1991; 11: 281-296Crossref PubMed Scopus (5311) Google Scholar) for various lengths of the C-terminal extension: a, full-length construct, 556–670; b, residues 556–666; c, residues 556–661; d, residues 556–656; e, nominal PH domain, residues 556–651. The βARK1 PH domain constructs in b–d correspond to C-terminal deletion studies of Gβγ binding (b and c (50Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar) and d (19Mahadevan D. Thanki N. Singh J. McPhie P. Zangrilli D. Wang L.M. Guerrero C. LeVine III, H. Humblet C. Saldanha J. Gutkind J.S. Najmabadi-Haske T. Biochemistry. 1995; 34: 9111-9117Crossref PubMed Scopus (62) Google Scholar)). The most C-terminal residues, upon truncation, are indicated. For comparison, the electrostatic potential of the PH domain from PLCδ (Protein Data Bank entry 1MAI) is shown in f; the arrow indicates a positively charged area at the opening of the β-barrel, which is involved in the phospholipid binding (8Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (522) Google Scholar). The molecular orientations are similar, as indicated by the backbone tube diagrams.View Large Image Figure ViewerDownload (PPT) Of specific note, the C-terminal α-helix is clearly extended by more than one turn compared with C-terminal α-helices of most previously determined PH domain structures. The position/orientation of the helix appears to be fixed by interactions with the protein core, namely by a hydrophobic strip formed by Leu-640, Trp-643, Leu-647, Ala-650, Tyr-651, and Ala-654, which are located on the side of the helix facing the β-sandwich and are involved in contacts with several residues in the first two β-strands. The aromatic ring of Trp-643, the only conserved residue among PH domains, is buried in the protein core and exhibits numerous NOE contacts to residues in β1 and β2. Another aromatic residue in the helix, Tyr-651, is also oriented toward the interior of the β-sandwich. The NOESY data indicate several close contacts between the aromatic ring of Tyr-651 and the residues in the β4/β5 loop and in the strand β5. Both the structure of this loop and the orientation of the Tyr-651 ring are well defined, as indicated by low r.m.s.d. values in these parts of the structure, and by chemical shift non-equivalence of all four ring hydrogens of Tyr-651. The hβARK1 PH domain has very low sequence similarity to other PH domains of known three-dimensional structure, and therefore cannot be satisfactorily homology-modeled from known structures. The structure-based alignment of the hβARK1 PH with these PH domains (TablesII and III) demonstrates the same overall topology of the protein fold. The expected range of r.m.s.d. values between sequences of the same structural class, but with varying degrees of homology, has been derived previously (33Chothia C. Lesk A.M. EMBO J. 1986; 5: 823-826Crossref PubMed Scopus (1947) Google Scholar). The r.m.s.d. values between different members of the PH/PTB domain family (Fig. 7) are within the range expected for such homologous sequences of low identity. The charge distribution is, however, different among the PH domains, and the large positive charge associated with the C-terminal helix of βARK is unusual. The low pH (4.5) required for this study is close to the pKa values for both glutamate and aspartate, so variations in the side chain charges of these residues compared with physiological conditions are expected. Since this might result in a perturbed structure in those regions containing negatively charged residues, a question arises of whether the NMR structure derived under these conditions represents the protein structure under physiological conditions. As mentioned above, the circular dichroism data indicate no changes in the protein structure as compared with the more physiological conditions in a phosphate-buffered saline pH 7.2. To address this issue in greater detail, the1H-15N correlation maps (HSQC) were also recorded for the PH domain dissolved in the phosphate-buffered saline (pH 6.0, temperature 25 °C) or in 0.1 m Tris buffer (pH 7.9, 35 °C). The minor chemical shift changes (up to 0.06 ppm in1H and 0.6 ppm in 15N) are consistent with expectations of variations in pH, temperature, and buffer content. The absence of significant chemical shift perturbations in this fingerprint region suggests no significant changes in the protein structure. The βARK PH domain tertiary structure here is also generally similar to other PH domain structures measured in the range pH 6.0–9.0 (8Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1995; 83: 1037-1046Abstract Full Text PDF PubMed Scopus (522) Google Scholar, 11Hyvonen M. Macias M.J. Nilges M. Oschkinat H. Saraste M. Wilmanns M. EMBO J. 1995; 14: 4676-4685Crossref PubMed Scopus (303) Google Scholar,34Fushman D. Cahill S. Lemmon M.A. Schlessinger J. Cowburn D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 816-820Crossref PubMed Scopus (82) Google Scholar, 35Ferguson K.M. Lemmon M.A. Schlessinger J. Sigler P.B. Cell. 1994; 79: 199-209Abstract Full Text PDF PubMed Scopus (240) Google Scholar, 36Macias M.J. Musacchio A. Ponstingl H. Nilges M. Saraste M. Oschkinat H. Nature. 1994; 369: 675-677Crossref PubMed Scopus (207) Google Scholar, 37Zhang P. Talluri S. Deng H. Branton D. Wagner G. Structure. 1995; 3: 1185-1195Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 38Yoon H.S. Hajduk P.J. Petros A.M. Olejniczak E.T. Meadows R.P. Fesik S.W. Nature. 1994; 371: 672-675Crossref Scopus (187) Google Scholar, 39Hyvonen M. Saraste M. EMBO J. 1997; 16: 3396-3404Crossref PubMed Scopus (189) Google Scholar). The high flexibility of the C terminus in the extended βARK PH domain construct reported here is also preserved at the more physiological conditions, as indicated by negative steady-state heteronuclear NOEs observed for the C-terminal residues (663–670) in phosphate-buffered saline (pH 6.0, 25 °C). The binding to Gβγ subunits is also retained at pH 4.5, with c. 100 nm affinity of the GST fusion protein at pH 4.5 and 7.5, from an immunoblotted Western assay (19Mahadevan D. Thanki N. Singh J. McPhie P. Zangrilli D. Wang L.M. Guerrero C. LeVine III, H. Humblet C. Saldanha J. Gutkind J.S. Najmabadi-Haske T. Biochemistry. 1995; 34: 9111-9117Crossref PubMed Scopus (62) Google Scholar). The C-terminal segment shows an unusual structural feature that, to our knowledge, has not been reported previously in proteins. NOEs characteristic for an α-helix are preserved for residues toward the C terminus despite the gradual loss of other NMR characteristics of helical structure (deviations from standard chemical shifts, heteronuclear 15N{1H} NOEs,3 JHNHα coupling) (Fig. 2). Increased mobility is indicated by both relaxation and solvent exchange/accessibility data, suggesting that the C-terminal part of the α-helix is present as a “molten helix” in solution. Molecular dynamics calculations (40Soman K.V. Karimi A. Case D.A. Biopolymers. 1991; 31: 1351-1361Crossref PubMed Scopus (138) Google Scholar) of α-helical melting appear to be qualitatively consistent with our NMR observations. The hydrophobic residues C-terminal to Gln-656 (Leu-657, Val-658, and Val-661) are located at proper sequence positions to extend the already existing hydrophobic strip on the helix surface. However, being extended beyond possible interaction" @default.
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