Phosphoprotein and phosphopeptide interactions with the FHA domain from Arabidopsis kinase-associated protein phosphatase

FHA domains are phosphoThr recognition modules found in diverse signaling proteins, including kinase-associated protein phosphatase (KAPP) from Arabidopsis thaliana. The kinase-interacting FHA domain (KI-FHA) of KAPP targets it to function as a negative regulator of some receptor-like kinase (RLK) signaling pathways important in plant development and environmental responses. To aid in the identification of potential binding sites for the KI-FHA domain, we predicted (i) the structure of a representative KAPP-binding RLK, CLAVATA1, and (ii) the functional surfaces of RLK kinase domains using evolutionary trace analysis. We selected phosphopeptides from KAPP-binding Arabidopsis RLKs for in vitro studies of association with KI-FHA from KAPP. Three phosphoThr peptide fragments from the kinase domain of CLV1 or BAK1 were found to bind KI-FHA with KD values of 8-20 microM, by NMR or titration calorimetry. Their affinity is driven by favorable enthalpy and solvation entropy gain. Mutagenesis of these three threonine sites suggests Thr546 in the C-lobe of the BAK1 kinase domain to be a principal but not sole site of KI-FHA binding in vitro. The brassinosteroid receptor BRI1 and KAPP are shown to associate in vivo and in vitro. Further genetic studies indicate that KAPP may be a negative regulator of the BRI1 signaling transduction pathway. 15N-Labeled KI-FHA was titrated with the GST-BRI1 kinase domain and monitored by NMR. BRI1 interacts with the same 3/4, 4/5, 6/7, 8/9, and 10/11 recognition loops of KI-FHA, with similar affinity as the phosphoThr peptides.     

Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9

Forkhead-associated (FHA) domains have been shown to recognize both pThr and pTyr-peptides. The solution structures of the FHA2 domain of Rad53 from Saccharomyces cerevisiae, and its complex with a pTyr peptide, have been reported recently. We now report the solution structure of the other FHA domain of Rad53, FHA1 (residues 14-164), and identification of binding sites of FHA1 and its target protein Rad9. The FHA1 structure consists of 11 beta-strands, which form two large twisted anti-parallel beta-sheets folding into a beta-sandwich. Three short alpha-helices were also identified. The beta-strands are linked by several loops and turns. These structural features of free FHA1 are similar to those of free FHA2, but there are significant differences in the loops. Screening of a peptide library [XXX(pT)XXX] against FHA1 revealed an absolute requirement for Asp at the +3 position and a preference for Ala at the +2 position. These two criteria are met by a pThr motif (192)TEAD(195) in Rad9. Surface plasmon resonance analysis showed that a pThr peptide containing this motif, (188)SLEV(pT)EADATFVQ(200) from Rad9, binds to FHA1 with a K(d) value of 0.36 microM. Other peptides containing pTXXD sequences also bound to FHA1, but less tightly (K(d)=4-70 microM). These results suggest that Thr192 of Rad9 is the likely phosphorylation site recognized by the FHA1 domain of Rad53. The tight-binding peptide was then used to identify residues of FHA1 involved in the interaction with the pThr peptide. The results are compared with the interactions between the FHA2 domain and a pTyr peptide derived from Rad9 reported previously.     

Achieving Peptide Binding Specificity and Promiscuity by Loops: Case of the Forkhead-Associated Domain

The regulation of a series of cellular events requires specific protein–protein interactions, which are usually mediated by modular domains to precisely select a particular sequence from diverse partners. However, most signaling domains can bind to more than one peptide sequence. How do proteins create promiscuity from precision? Moreover, these complex interactions typically occur at the interface of a well-defined secondary structure, α helix and β sheet. However, the molecular recognition primarily controlled by loop architecture is not fully understood. To gain a deep understanding of binding selectivity and promiscuity by the conformation of loops, we chose the forkhead-associated (FHA) domain as our model system. The domain can bind to diverse peptides via various loops but only interact with sequences containing phosphothreonine (pThr). We applied molecular dynamics (MD) simulations for multiple free and bound FHA domains to study the changes in conformations and dynamics. Generally, FHA domains share a similar folding structure whereby the backbone holds the overall geometry and the variety of sidechain atoms of multiple loops creates a binding surface to target a specific partner. FHA domains determine the specificity of pThr by well-organized binding loops, which are rigid to define a phospho recognition site. The broad range of peptide recognition can be attributed to different arrangements of the loop interaction network. The moderate flexibility of the loop conformation can help access or exclude binding partners. Our work provides insights into molecular recognition in terms of binding specificity and promiscuity and helpful clues for further peptide design.     

Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53

The forkhead-associated (FHA) domain is a 55-75 amino acid residue module found in >20 proteins from yeast to human. It has been suggested to participate in signal transduction pathways, perhaps via protein-protein interactions involving recognition of phosphopeptides. Neither the structure nor the ligand of FHA is known. Yeast Rad53, a checkpoint protein involved in DNA damage response, contains two FHA domains, FHA1 (residues 66-116) and FHA2 (residues 601-664), the second of which recognizes phosphorylated Rad9. We herein report the solution structure of an "FHA2-containing domain" of Rad53 (residues 573-730). The structure consists of a beta-sandwich containing two antiparallel beta-sheets and a short, C-terminal alpha-helix. Binding experiments suggested that the FHA2-containing domain specifically recognizes pTyr and a pTyr-containing peptide from Rad9, and that the binding site involves residues highly conserved across FHA domains. The results, along with other recent reports, suggest that FHA domains could have pTyr and pSer/Thr dual specificity.     

Solution structure of the yeast Rad53 FHA2 complexed with a phosphothreonine peptide pTXXL: comparison with the structures of FHA2-pYXL and FHA1-pTXXD complexes

It was proposed previously that the FHA2 domain of the yeast protein kinase Rad53 has dual specificity toward pY and pT peptides. The consensus sequences of pY peptides for binding to FHA2, as well as the solution structures of free FHA2 and FHA2 complex with a pY peptide derived from Rad9, have been obtained previously. We now report the use of a pT library to screen for binding of pT peptides with the FHA2 domain. The results show that FHA2 binds favorably to pT peptides with Ile at the +3 position. We then searched the Rad9 sequences with a pTXXI/L motif, and tested the binding affinity of FHA2 toward ten pT peptides derived from Rad9. One of the peptides, (599)EVEL(pT)QELP(607), displayed the best binding affinity (K(d)=12.9 microM) and the greatest chemical shift changes. The structure of the FHA2 complex with this peptide was then determined by solution NMR and the structure of the complex between FHA2 and the pY peptide (826)EDI(pY)YLD(832) was further refined. Structural comparison of these two complexes indicates that the Leu residue at the +3 position in the pT peptide and that at the +2 position in the pY peptide occupy a very similar position relative to the binding site residues from FHA2. This can explain why FHA2 is able to bind both pT and pY peptides. This position change from +3 to +2 could be the consequence of the size difference between Thr and Tyr. Further insight into the structural basis of ligand specificity of FHA domains was obtained by comparing the structures of the FHA2-pTXXL complex obtained in this work and the FHA1-pTXXD complex reported in the accompanying paper.     

Partially unfolded forms and non-two-state folding of a beta-sandwich: FHA domain from Arabidopsis receptor kinase-associated protein phosphatase

FHA domains adopt a beta-sandwich fold with 11 strands. The first evidence of partially unfolded forms of a beta-sandwich is derived from native-state hydrogen exchange (NHX) of the forkhead-associated (FHA) domain from kinase-associated protein phosphatase from Arabidopsis. The folding kinetics of this FHA domain indicate that EX2 behavior prevails at pH 6.3. In the chevron plot, rollover in the folding arm and bends in the unfolding arm suggest folding intermediates. NHX of this FHA domain suggests a core of six most stable beta-strands and two loops, characterized by rare global unfolding events. Flanking this stable core are beta-strands and recognition loops with less stability, termed subglobal motifs. These suggest partially unfolded forms (near-native intermediates) with two levels of stability. The spatial separation of the subglobal motifs on the flanks suggests possible parallelism in their folding as additional beta-strands align with the stable core of six strands. Intermediates may contribute to differences in stabilities and m-values suggested by NHX or kinetics relative to chemical denaturation. Residual structure in the unfolded regime is suggested by superprotection of beta-strand 6 and by GdmCl-dependence of adjustments in amide NMR spectra and residual optical signal. The global folding stability depends strongly on pH, with at least 3 kcal/mol more stability at pH 7.3 than at pH 6.3. This FHA domain is hypothesized to fold progressively with initial hydrophobic collapse of its stable six-stranded core followed by addition of less stable flanking beta-strands and ordering of recognition loops.     

Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism

To elucidate the influence of local motion of the polypeptide chain on the catalytic mechanism of an enzyme, we have measured (15)N relaxation data for Escherichia coli dihydrofolate reductase in three different complexes, representing different stages in the catalytic cycle of the enzyme. NMR relaxation data were analyzed by the model-free approach, corrected for rotational anisotropy, to provide insights into the backbone dynamics. There are significant differences in the backbone dynamics in the different complexes. Complexes in which the cofactor binding site is occluded by the Met20 loop display large amplitude motions on the picosecond/nanosecond time scale for residues in the Met20 loop, the adjacent betaF-betaG loop and for residues 67-69 in the adenosine binding loop. Formation of the closed Met20 loop conformation in the ternary complex with folate and NADP(+), results in attenuation of the motions in the Met20 loop and the betaF-betaG loop but leads to increased flexibility in the adenosine binding loop. New fluctuations on a microsecond/millisecond time scale are observed in the closed E:folate:NADP(+) complex in regions that form hydrogen bonds between the Met20 and the betaF-betaG loops. The data provide insights into the changes in backbone dynamics during the catalytic cycle and point to an important role of the Met20 and betaF-betaG loops in controlling access to the active site. The high flexibility of these loops in the occluded conformation is expected to promote tetrahydrofolate-assisted product release and facilitate binding of the nicotinamide ring to form the Michaelis complex. The backbone fluctuations in the Met20 loop become attenuated once it closes over the active site, thereby stabilizing the nicotinamide ring in a geometry conducive to hydride transfer. Finally, the relaxation data provide evidence for long-range motional coupling between the adenosine binding loop and distant regions of the protein.     

Exploring the potential of the monobody scaffold: effects of loop elongation on the stability of a fibronectin type III domain

The tenth fibronectin type III domain of human fibronectin (FNfn10) is a small, monomeric beta-sandwich protein, similar to the immunoglobulins. We have developed small antibody mimics, 'monobodies', using FNfn10 as a scaffold. We initially altered two loops of FNfn10 that are structurally equivalent to two of the hypervariable loops of the immunoglobulin domain. In order to assess the possibility of utilizing other loops in FNfn10 for target binding, we determined the effects of the elongation of each loop on the conformational stability of FNfn10. We found that all six loops of FNfn10 allowed the introduction of four glycine residues while retaining the global fold. Insertions in the AB and FG loops exhibited very small degrees of destabilization, comparable to or less than predicted entropic penalties due to the elongation, suggesting the absence of stabilizing interactions in these loops in wild-type FNfn10. Insertions in the BC, CD and DE loops, respectively, resulted in modest destabilization. In contrast, the EF loop elongation was highly destabilizing, consistent with previous studies showing the presence of stabilizing interactions in this loop. These results suggest that all loops, except for the EF loop, can be used for engineering a binding site, thus demonstrating excellent properties of the monobody scaffold.     

Crystal Structure of the Pml1p Subunit of the Yeast Precursor mRNA Retention and Splicing Complex

The precursor mRNA retention and splicing (RES) complex mediates nuclear retention and enhances splicing of precursor mRNAs. The RES complex from yeast comprises three proteins, Snu17p, Bud13p and Pml1p. Snu17p acts as a central platform that concomitantly binds the Bud13p and Pml1p subunits via short peptide epitopes. As a step to decipher the molecular architecture of the RES complex, we have determined crystal structures of full-length Pml1p and N-terminally truncated Pml1p. The first 50 residues of full-length Pml1p, encompassing the Snu17p-binding region, are disordered, showing that Pml1p binds to Snu17p via an intrinsically unstructured region. The remainder of Pml1p folds as a forkhead-associated (FHA) domain, which is expanded by a number of noncanonical elements compared with known FHA domains from other proteins. An atypical N-terminal appendix runs across one β-sheet and thereby stabilizes the domain as shown by deletion experiments. FHA domains are thought to constitute phosphopeptide-binding elements. Consistently, a sulfate ion was found at the putative phosphopeptide-binding loops of full-length Pml1p. The N-terminally truncated version of the protein lacked a similar phosphopeptide mimic but retained an almost identical structure. A long loop neighboring the putative phosphopeptide-binding site was disordered in both structures. Comparison with other FHA domain proteins suggests that this loop adopts a defined conformation upon ligand binding and thereby confers ligand specificity. Our results show that in the RES complex, an FHA domain of Pml1p is flexibly tethered via an unstructured N-terminal region to Snu17p.     

Structure of human Ki67 FHA domain and its binding to a phosphoprotein fragment from hNIFK reveal unique recognition sites and new views to the structural basis of FHA domain functions

Recent studies by use of short phosphopeptides showed that forkhead-associated (FHA) domains recognize pTXX(D/I/L) motifs. Solution structures and crystal structures of several different FHA domains and their complexes with short phosphopeptides have been reported by several groups. We now report the solution structure of the FHA domain of human Ki67, a large nuclear protein associated with the cell-cycle. Using fragments of its binding partner hNIFK, we show that Ki67-hNIFK binding involves ca 44 residues without a pTXX(D/I/L) motif. The pThr site of hNIFK recognized by Ki67 FHA is pThr234-Pro235, a motif also recognized by the proline isomerase Pin1. Heteronuclear single quantum coherence (HSQC) NMR was then used to map out the binding surface, and structural analyses were used to identify key binding residues of Ki67 FHA. The results represent the first structural characterization of the complex of an FHA domain with a biologically relevant target protein fragment. Detailed analyses of the results led us to propose that three major factors control the interaction of FHA with its target protein: the pT residue, +1 to +3 residues, and an extended binding surface, and that variation in the three factors is the likely cause of the great diversity in the function and specificity of FHA domains from different sources.     

Mapping by site-directed mutagenesis of the region responsible for cohesin-dockerin interaction on the surface of the seventh cohesin domain of Clostridium thermocellum CipA

To locate the region involved in binding dockerin domains, 15 mutations were introduced across the surface of the seventh cohesin domain of the scaffolding protein CipA, which holds together the cellulosome of Clostridium thermocellum. Mutated residues were located on both faces of the nine-stranded beta-sandwich forming the cohesin domain and on the loops connecting beta-strands 4 and 5, 6 and 7, and 8 and 9. The loop region was previously proposed, on the basis of sequence comparisons, to form a contiguous "recognition strip". Individual mutants of four residues, D39, Y74, E86, and G89, formed no complexes detectable by nondenaturing gel electrophoresis after incubation with CelD664, a shortened form of endoglucanase CelD lacking the residues linking the catalytic domain with the dockerin domain. The four sensitive residues encompass a hydrophobic region on the 5-6-3-8 face of the molecule, which overlaps partially with the recognition strip and with a hydrophobic zone involved in the formation of cohesin-cohesin dimers. Isothermal titration calorimetry showed that single cohesin mutations affecting the binding of CelD664 had significant effects on the enthalpy or entropy of binding of wild-type CelD but much lesser effects on the association constant, owing to enthalpy-entropy compensation. However, the affinity for wild-type CelD of the triple mutant affecting D39, Y74, and E86 was reduced by 2 orders of magnitude, due to negative cooperativity between mutations affecting D39 + Y74 on one hand and E86 on the other hand.     

The ligand specificity of yeast Rad53 FHA domains at the +3 position is determined by nonconserved residues

On the basis of the results from our laboratory and others, we recently suggested that the ligand specificity of forkhead-associated (FHA) domains is controlled by variations in three major factors: (i) residues interacting with pThr, (ii) residues recognizing the +1 to +3 residues from pThr, and (iii) an extended binding surface. While the first factor has been well established by several solution and crystal structures of FHA-phosphopeptide complexes, the structural bases of the second and third factors are not well understood and are likely to vary greatly between different FHA domains. In this work, we proposed and tested the hypothesis that nonconserved residues G133 and G135 of FHA1 and I681 and D683 of FHA2, located outside of the core FHA region of yeast Rad53 FHA domains, contribute to the specific recognition of the +3 position of different phosphopeptides. By rational mutagenesis of these residues, the specificity of FHA1 has been changed from predominantly pTXXD to be equally acceptable for pTXXD, pTXXL, and pYXL, which are similar to the specificities of the FHA2 domain of Rad53. Conversely, the +3 position specificity of FHA2 has been engineered to be more like FHA1 with the I681A mutation. These results were based on library screening as well as binding analyses of specific phosphopeptides. Furthermore, results of structural analyses by NMR indicate that some of these residues are also important for the structural integrity of the loops.     

Structural and functional analysis of phosphothreonine-dependent FHA domain interactions

FHA domains are well established as phospho-dependent binding modules mediating signal transduction in Ser/Thr kinase signaling networks in both eukaryotic and prokaryotic species. Although they are unique in binding exclusively to phosphothreonine, the basis for this discrimination over phosphoserine has remained elusive. Here, we attempt to dissect overall binding specificity at the molecular level. We first determined the optimal peptide sequence for Rv0020c FHA domain binding by oriented peptide library screening. This served as a basis for systematic mutagenic and binding analyses, allowing us to derive relative thermodynamic contributions of conserved protein and peptide residues to binding and specificity. Structures of phosphopeptide-bound and uncomplexed Rv0020c FHA domain then directed molecular dynamics simulations which show how the extraordinary discrimination in favor of phosphothreonine occurs through formation of additional hydrogen-bonding networks that are ultimately stabilized by van der Waals interactions of the phosphothreonine γ-methyl group with a conserved pocket on the FHA domain surface.     

Tissue inhibitor of metalloproteinases-1 undergoes microsecond to millisecond motions at sites of matrix metalloproteinase-induced fit

The N-terminal, matrix metalloproteinase (MMP)-inhibitory fragment of recombinant, human tissue inhibitor of metalloproteinases (TIMP-1) exhibits varied backbone dynamics and rigidity. Most striking is the presence of chemical exchange in the MMP-binding ridge reported to undergo conformational change upon MMP binding. Conformational exchange fluctuations in microseconds to milliseconds map to the sites of MMP-induced fit at residues Val29 through Leu34 of the AB loop and to the Ala65 and Cys70 "hinges" of the CD loop of TIMP-1. Slow chemical exchange is also present at the type I turn of the EF loop at the base of the MMP-binding ridge. These functional slow motions and other fast internal motions are evident from backbone (15)N spin relaxation at 500 and 750 MHz, whether interpreted by the model-free formalism with axial diffusion anisotropy or by the reduced spectral density approach. The conformational exchange is confirmed by its deviation from the trend between R(2) and the cross-correlation rate eta. The magnetic field-dependence indicates that the chemical exchange broadening in the AB and CD loops is fast on the time-scale of chemical shift differences. The conformational exchange rates for most of these exchanging residues, which can closely approach MMP, appear to be a few thousand to several thousand per second. The slow dynamics of the TIMP-1 AB loop contrast the picosecond to nanosecond dynamics reported in the longer TIMP-2 AB loop.     

Solution structure of the PDZ2 domain from human phosphatase hPTP1E and its interactions with C-terminal peptides from the Fas receptor

The solution structure of the second PDZ domain (PDZ2) from human phosphatase hPTP1E has been determined using 2D and 3D heteronuclear NMR experiments. The binding of peptides derived from the C-terminus of the Fas receptor to PDZ2 was studied via changes in backbone peptide and protein resonances. The structure is based on a total of 1387 nonredundant experimental NMR restraints including 1261 interproton distance restraints, 45 backbone hydrogen bonds, and 81 torsion angle restraints. Analysis of 30 lowest-energy structures resulted in rmsd values of 0.41 +/- 0.09 A for backbone atoms (N, Calpha, C') and 1.08 +/- 0.10 A for all heavy atoms, excluding the disordered N- and C-termini. The hPTP1E PDZ2 structure is similar to known PDZ domain structures but contains two unique structural features. In the peptide binding domain, the first glycine of the GLGF motif is replaced by a serine. This serine appears to replace a bound water observed in PDZ crystal structures that hydrogen bonds to the bound peptide's C-terminus. The hPTP1E PDZ2 structure also contains an unusually large loop following strand beta2 and proximal to the peptide binding site. This well-ordered loop folds back against the PDZ domain and contains several residues that undergo large amide chemical shift changes upon peptide binding. Direct observation of peptide resonances demonstrates that as many as six Fas peptide residues interact with the PDZ2 domain.     

Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain

MDC1 is a key mediator of the DNA-damage response in mammals with several phosphorylation-dependent protein interaction domains. The function of its N-terminal forkhead-associated (FHA) domain remains elusive. Here, we show with structural, biochemical and cellular data that the FHA domain mediates phosphorylation-dependent dimerization of MDC1 in response to DNA damage. Crystal structures of the FHA domain reveal a face-to-face dimer with pseudo-dyad symmetry. We found that the FHA domain recognizes phosphothreonine 4 (pT4) at the N-terminus of MDC1 and determined its crystal structure in complex with a pT4 peptide. Biochemical analysis further revealed that in the dimer, the FHA domain binds in trans to pT4 from the other subunit, which greatly stabilizes the otherwise unstable dimer. We show that T4 is phosphorylated primarily by ATM upon DNA damage. MDC1 mutants with the FHA domain deleted or impaired in its ability to dimerize formed fewer foci at DNA-damage sites, but the localization defect was largely rescued by an artificial dimerization module, suggesting that dimerization is the primary function of the MDC1 FHA domain. Our results suggest a novel mechanism for the regulation of MDC1 function through T4 phosphorylation and FHA-mediated dimerization.     

Directed discovery of bivalent peptide ligands to an SH3 domain

The Caenorhabditis elegans SEM-5 SH3 domains recognize proline-rich peptide segments with modest affinity. We developed a bivalent peptide ligand that contains a naturally occurring proline-rich binding sequence, tethered by a glycine linker to a disulfide-closed loop segment containing six variable residues. The glycine linker allows the loop segment to explore regions of greatest diversity in sequence and structure of the SH3 domain: the RT and n-Src loops. The bivalent ligand was optimized using phage display, leading to a peptide (PP-G(4)-L) with 1000-fold increased affinity for the SEM-5 C-terminal SH3 domain over that of a natural ligand. NMR analysis of the complex confirms that the peptide loop segment is targeted to the RT and n-Src loops and parts of the beta-sheet scaffold of this SH3 domain. This binding region is comparable to that targeted by a natural non-PXXP peptide to the p67(phox) SH3 domain, a region not known to be targeted in the Grb2 SH3 domain family. PP-G(4)-L may aid in the discovery of additional binding partners of Grb2 family SH3 domains.     

NMR solution structure of the receptor binding domain of human alpha(2)-macroglobulin

Human alpha(2)-macroglobulin-proteinase complexes bind to their receptor, the low density lipoprotein receptor-related protein (LRP), through a discrete 138-residue C-terminal receptor binding domain (RBD), which also binds to the beta-amyloid peptide. We have used NMR spectroscopy on recombinantly expressed uniformly (13)C/(15)N-labeled human RBD to determine its three-dimensional structure in solution. Human RBD is a sandwich of two antiparallel beta-sheets, one four-strand and one five-strand, and also contains one alpha-helix of 2.5 turns and an additional 1-turn helical region. The principal alpha-helix contains two lysine residues on the outer face that are known to be essential for receptor binding. A calcium binding site (K(d) approximately 11 mM) is present in the loop region at one end of the beta-sandwich. Calcium binding principally affects this loop region and does not significantly perturb the stable core structure of the domain. The structure and NMR assignments will enable us to examine in solution specific binding of RBD to domains of the receptor and to beta-amyloid peptide.     

Peptide design and structural characterization of a GPCR loop mimetic

G protein-coupled receptors (GPCRs) control fundamental aspects of human physiology and behaviors. Knowledge of their structures, especially for the loop regions, is limited and has principally been obtained from homology models, mutagenesis data, low resolution structural studies, and high resolution studies of peptide models of receptor segments. We developed an alternate methodology for structurally characterizing GPCR loops, using the human S1P(4) first extracellular loop (E1) as a model system. This methodology uses computational peptide designs based on transmembrane domain (TM) model structures in combination with CD and NMR spectroscopy. The characterized peptides contain segments that mimic the self-assembling extracellular ends of TM 2 and TM 3 separated by E1, including residues R3.28(121) and E3.29(122) that are required for sphingosine 1-phosphate (S1P) binding and receptor activation in the S1P(4) receptor. The S1P(4) loop mimetic peptide interacted specifically with an S1P headgroup analog, O-phosphoethanolamine (PEA), as evidenced by PEA-induced perturbation of disulfide cross-linked coiled-coil first extracellular loop mimetic (CCE1a) (1)H and (15)N backbone amide chemical shifts. CCE1a was capable of weakly binding PEA near biologically relevant residues R29 and E30, which correspond to R3.28 and E3.29 in the full-length S1P(4) receptor, confirming that it has adopted a biologically relevant conformation. We propose that the combination of coiled-coil TM replacement and conformational stabilization with an interhelical disulfide bond is a general design strategy that promotes native-like structure for loops derived from GPCRs.     

Membrane-bound orientation and position of the synaptotagmin I C2A domain by site-directed spin labeling

Site-directed spin labeling was used to determine the membrane orientation and insertion of the C2A domain from synaptotagmin I. A series of single cysteine mutants of the C2A domain of synaptotagmin I was prepared and labeled with a sulfhydryl specific spin label. Upon Ca2+ or membrane binding, the EPR line shapes of these mutants reveal dramatic decreases in label mobility within the Ca2+-binding loops. This loss in mobility is likely due in part to a reduction in local backbone fluctuations within the loop regions. Power saturation was then used to determine the position of each spin-labeled site along the bilayer normal, and these EPR distance constraints were used along with the high-resolution solution structure of C2A to generate a model for the orientation and position of the domain at the membrane interface. This model places the polypeptide backbone of both the first and third Ca2+-binding loops in contact with the membrane interface, with several labeled side chains lying within the bilayer interior. All three Ca2+-binding sites lie near a plane defined by the lipid phosphates. This model indicates that there is some desolvation of this domain upon binding and that hydrophobic as well as electrostatic interactions contribute to the binding of C2A. When compared to the C2 domain from cPLA2 (Frazier et al. (2002) Biochemistry 41, 6282), a similar orientation for the beta-sandwich region is found; however, the cPLA2 C2 domain is translocated 5-7 A deeper into the membrane hydrocarbon. This difference in depth is consistent with previous biophysical data and with the difference that long-range electrostatic interactions and desolvation are expected to make to the binding of these two C2 domains.