Xaa-Arg-Gly triplets in the collagen triple helix are dominant binding sites for the molecular chaperone HSP47.

HSP47 is an essential procollagen-specific molecular chaperone that resides in the endoplasmic reticulum of procollagen-producing cells. Recent advances have revealed that HSP47 recognizes the (Pro-Pro-Gly)(n) sequence but not (Pro-Hyp-Gly)(n) and that HSP47 recognizes the triple-helical conformation. In this study, to better understand the substrate recognition by HSP47, we synthesized various collagen model peptides and examined their interaction with HSP47 in vitro. We found that the Pro-Arg-Gly triplet forms an HSP47-binding site. The HSP47 binding was observed only when Arg residues were incorporated in the Yaa positions of the Xaa-Yaa-Gly triplets. Amino acids in the Xaa position did not largely affect the interaction. The recognition of the Arg residue by HSP47 was specific to its side-chain structure because replacement of the Arg residue by other basic amino acids decreased the affinity to HSP47. The significance of Arg residues in HSP47 binding was further confirmed by using residue-specific chemical modification of types I and III collagen. Our results demonstrate that Xaa-Arg-Gly sequences in the triple-helical procollagen molecule are dominant binding sites for HSP47 and enable us to predict HSP47-binding sites in homotrimeric procollagen molecules.     

Substrate recognition of collagen-specific molecular chaperone HSP47. Structural requirements and binding regulation

Prior to secretion, procollagen molecules are correctly folded to triple helices in the endoplasmic reticulum (ER). HSP47 specifically associates with procollagen in the ER during its folding and/or modification processes and is thought to function as a collagen-specific molecular chaperone (Nagata, K. (1996) Trends Biochem. Sci. 21, 23-26). However, structural requirements for substrate recognition and regulation of the binding have not yet been elucidated. Here, we show that a typical collagen model sequence, (Pro-Pro-Gly)(n), possesses sufficient structural information required for recognition by HSP47. A structure-activity relationship study using synthetic analogs of (Pro-Pro-Gly)(n) has revealed the requirements in both chain length and primary structure for the interaction. The substrate recognition of HSP47 has also been shown to be similar but distinct from that of prolyl 4-hydroxylase, an ER resident enzyme. Further, it has shown that the interaction of HSP47 with the substrate peptides is abolished by prolyl 4-hydroxylation of the second Pro residues in Pro-Pro-Gly triplets and that the fully prolyl 4-hydroxylated peptide, (Pro-Hyp-Gly)(n), does not interact with HSP47. We thus have proposed a model in which HSP47 dissociates from procollagen during the process of prolyl 4-hydroxylation in the ER.     

Collagen’s primary structure determines collagen:HSP47 complex stoichiometry

Collagens play important roles in development and homeostasis in most higher organisms. In order to function, collagens require the specific chaperone HSP47 for proper folding and secretion. HSP47 is known to bind to the collagen triple helix, but the exact positions and numbers of binding sites are not clear. Here, we employed a collagen II peptide library to characterize high-affinity binding sites for HSP47. We show that many previously predicted binding sites have very low affinities due to the presence of a negatively charged amino acid in the binding motif. In contrast, large hydrophobic amino acids such as phenylalanine at certain positions in the collagen sequence increase binding strength. For further characterization, we determined two crystal structures of HSP47 bound to peptides containing phenylalanine or leucine. These structures deviate significantly from previously published ones in which different collagen sequences were used. They reveal local conformational rearrangements of HSP47 at the binding site to accommodate the large hydrophobic side chain from the middle strand of the collagen triple helix and, most surprisingly, possess an altered binding stoichiometry in the form of a 1:1 complex. This altered stoichiometry is explained by steric collisions with the second HSP47 molecule present in all structures determined thus far caused by the newly introduced large hydrophobic residue placed on the trailing strand. This exemplifies the importance of considering all three sites of homotrimeric collagen as independent interaction surfaces and may provide insight into the formation of higher oligomeric complexes at promiscuous collagen-binding sites.     

Specific recognition of the collagen triple helix by chaperone HSP47. II. The HSP47-binding structural motif in collagens and related proteins

The endoplasmic reticulum-resident chaperone heat-shock protein 47 (HSP47) plays an essential role in procollagen biosynthesis. The function of HSP47 relies on its specific interaction with correctly folded triple-helical regions comprised of Gly-Xaa-Yaa repeats, and Arg residues at Yaa positions have been shown to be important for this interaction. The amino acid at the Yaa position (Yaa(-3)) in the N-terminal-adjoining triplet containing the critical Arg (defined as Arg(0)) was also suggested to be directly recognized by HSP47 (Koide, T., Asada, S., Takahara, Y., Nishikawa, Y., Nagata, K., and Kitagawa, K. (2006) J. Biol. Chem. 281, 3432-3438). Based on this finding, we examined the relationship between the structure of Yaa(-3) and HSP47 binding using synthetic collagenous peptides. The results obtained indicated that the structure of Yaa(-3) determined the binding affinity for HSP47. Maximal binding was observed when Yaa(-3) was Thr. Moreover, the required relative spatial arrangement of these key residues in the triple helix was analyzed by taking advantage of heterotrimeric collagen-model peptides, each of which contains one Thr(-3) and one Arg(0). The results revealed that HSP47 recognizes the Yaa(-3) and Arg(0) residues only when they are on the same peptide strand. Taken together, the data obtained led us to define the HSP47-binding structural epitope in the collagen triple helix and also define the HSP47-binding motif in the primary structure. A motif search against human protein database predicted candidate clients for this molecular chaperone. The search result indicated that not all collagen family proteins require the chaperoning by HSP47.     

Specific recognition of the collagen triple helix by chaperone HSP47: minimal structural requirement and spatial molecular orientation

The unique folding of procollagens in the endoplasmic reticulum is achieved with the assistance of procollagen-specific molecular chaperones. Heat-shock protein 47 (HSP47) is an endoplasmic reticulum-resident chaperone that plays an essential role in normal procollagen folding, although its molecular function has not yet been clarified. Recent advances in studies on the binding specificity of HSP47 have revealed that Arg residues at Yaa positions in collagenous Gly-Xaa-Yaa repeats are critical for its interactions (Koide, T., Takahara, Y., Asada, S., and Nagata, K. (2002) J. Biol. Chem. 277, 6178-6182; Tasab, M., Jenkinson, L., and Bulleid, N. J. (2002) J. Biol. Chem. 277, 35007-35012). In the present study, we further examined the client recognition mechanism of HSP47 by taking advantage of systems employing engineered collagen model peptides. First, in vitro binding studies using conformationally constrained collagen-like peptides revealed that HSP47 only recognized correctly folded triple helices and that the interaction with the corresponding single-chain polypeptides was negligible. Second, a binding study using heterotrimeric model clients for HSP47 demonstrated a minimal requirement for the number of Arg residues in the triple helix. Finally, a cross-linking study using photoreactive collagenous peptides provided information about the spatial orientation of an HSP47 molecule in the chaperone-collagen complex. The obtained results led to the development of a new model of HSP47-collagen complexes that differs completely from the previously proposed "flying capstan model" (Dafforn, T. R., Della, M., and Miller, A. D. (2001) J. Biol. Chem. 276, 49310-49319).     

The molecular interactions of heat shock protein 47 (Hsp47) and their implications for collagen biosynthesis

Heat shock protein 47 (Hsp47) is a procollagen/collagen-specific molecular chaperone protein derived from the serpin family of proteins and essential for the early stages of collagen biosynthesis. In this paper, the results of an extensive biophysical analysis of mature recombinant mouse Hsp47 show the existence of both a structurally mesostable monomer with a 5-strand A-sheet and/or a hyperstable trimer; both states have biological activity. It is also demonstrated that Hsp47 is able to bind to a monomeric and partially folded conformation collagen mimic peptide (PPG)(10). Upon peptide binding Hsp47 has the capacity to induce the peptide backbone to fold into a polyproline type II conformation. Induction of this conformation results in (PPG)(10) peptides associating into higher order assemblies with increased stability compared with the monomeric peptide alone. These assemblies are similar to those observed by others (Go, N., and Suezaki, Y. (1973) Biopolymers 12, 1927-1930; Engel, J., Chen, H. T., Prockop, D. J., and Klump, H. (1977) Biopolymers 16, 601-622) when the peptide is dissolved at high concentration and are proposed to be long chains of peptides in a collagen-like configuration. Examination of the biophysical characteristics of both monomeric and trimeric Hsp47-(PPG)(10) complexes is also used to determine that the peptide-binding site does not reside in strand 4 position of sheet A, as observed for other serpins (Skinner, R., Chang, W. S. W., Jin, L., Pei, X., Huntington, J. A., Abrahams, J. P., Carrell, R. W., and Lomas, D. A. (1998) J. Mol. Biol. 283, 9-14), leaving earlier proposals of a binding site near helix A viable.     

The Recognition of Collagen and Triple-helical Toolkit Peptides by MMP-13: SEQUENCE SPECIFICITY FOR BINDING AND CLEAVAGE*

Remodeling of collagen by matrix metalloproteinases (MMPs) is crucial to tissue homeostasis and repair. MMP-13 is a collagenase with a substrate preference for collagen II over collagens I and III. It recognizes a specific, well-known site in the tropocollagen molecule where its binding locally perturbs the triple helix, allowing the catalytic domain of the active enzyme to cleave the collagen α chains sequentially, at Gly⁷⁷⁵–Leu⁷⁷⁶ in collagen II. However, the specific residues upon which collagen recognition depends within and surrounding this locus have not been systematically mapped. Using our triple-helical peptide Collagen Toolkit libraries in solid-phase binding assays, we found that MMP-13 shows little affinity for Collagen Toolkit III, but binds selectively to two triple-helical peptides of Toolkit II. We have identified the residues required for the adhesion of both proMMP-13 and MMP-13 to one of these, Toolkit peptide II-44, which contains the canonical collagenase cleavage site. MMP-13 was unable to bind to a linear peptide of the same sequence as II-44. We also discovered a second binding site near the N terminus of collagen II (starting at helix residue 127) in Toolkit peptide II-8. The pattern of binding of the free hemopexin domain of MMP-13 was similar to that of the full-length enzyme, but the free catalytic subunit bound none of our peptides. The susceptibility of Toolkit peptides to proteolysis in solution was independent of the very specific recognition of immobilized peptides by MMP-13; the enzyme proved able to cleave a range of dissolved collagen peptides.     

Mapping Hsp47 binding site(s) using CNBr peptides derived from type I and type II collagen

As a crucial molecular chaperone in collagen biosynthesis, Hsp47 interacts with the nascent form as well as the mature triple-helical form of procollagen. The location(s) of Hsp47 binding sites on the collagen molecule are, as yet, unknown. We have examined the substrate specificity of Hsp47 in vitro using well-characterized CNBr peptide fragments of type I and type II collagen along with radiolabeled, recombinant Hsp47. Interaction of these peptides with Hsp47 bound to collagen-coated microtiter wells showed several binding sites for Hsp47 along the length of the alpha1 and alpha2 chains of type I collagen and the alpha1 chain of type II collagen, with the N-terminal regions showing the strongest affinities. The latter observation was also supported by the results of a ligand-blot assay. Except for two peptides in the alpha2(I) chain, peptides that showed substantial binding to Hsp47 did so in their triple-helical and not random-coil form. Unlike earlier studies that used peptide models for collagen, the results obtained here on fragments of type I and type II collagen identify, for the first time, binding of Hsp47 to specific regions of the collagen molecule. They also point to additional structural requirements for Hsp47 binding besides the known preference for third-position Arg residues and the triple-helical conformation.     

Phagotopes derived by antibody screening of phage-displayed random peptide libraries vary in immunoreactivity: studies using an exemplary monoclonal antibody, CII-C1, to type II collagen

Antibody screening of phage-displayed random peptide libraries to identify mimotopes of conformational epitopes is promising. However, because interpretations can be difficult, an exemplary system has been used in the present study to investigate whether variation in the peptide sequences of selected phagotopes corresponded with variation in immunoreactivity. The phagotopes, derived using a well-characterized monoclonal antibody, CII-C1, to a known conformational epitope on type II collagen, C1, were tested by direct and inhibition ELISA for reactivity with CII-C1. A multiple sequence alignment algorithm, PILEUP, was used to sort the peptides expressed by the phagotopes into clusters. A model was prepared of the C1 epitope on type II collagen. The 12 selected phagotopes reacted with CII-C1 by both direct ELISA (titres from < 100-11 200) and inhibition ELISA (20-100% inhibition); the reactivity varied according to the peptide sequence and assay format. The differences in reactivity between the phagotopes were mostly in accord with the alignment, by PILEUP, of the peptide sequences. The finding that the phagotopes functionally mimicked the C1 epitope on collagen was validated in that amino acids RRL at the amino terminal of many of the peptides were topographically demonstrable on the model of the C1 epitope. Notably, one phagotope that expressed the widely divergent peptide C-IAPKRHNSA-C also mimicked the C1 epitope, as judged by reactivity in each of the assays used: these included cross-inhibition of CII-C1 reactivity with each of the other phagotopes and inhibition by a synthetic peptide corresponding to that expressed by the most frequently selected phagotope, RRLPFGSQM. Thus, it has been demonstrated that multiple phage-displayed peptides can mimic the same epitope and that observed immunoreactivity of selected phagotopes with the selecting mAb can depend on the primary sequence of the expressed peptide and also on the assay format used.     

HSP47 binds cooperatively to triple helical type I collagen but has little effect on the thermal stability or rate of refolding

HSP47, a collagen-specific molecular chaperone, interacts with unfolded and folded procollagens. Binding of chicken HSP47 to native bovine type I collagen was studied by fluorescence quenching and cooperative binding with a collagen concentration at half saturation (K(half)) of 1.4 x 10(-7) m, and a Hill coefficient of 4.3 was observed. Similar results are observed for the binding of mouse HSP47 recombinantly expressed in Escherichia coli. Chicken HSP47 binds equally well to native type II and type III procollagen without the carboxyl-terminal propeptide (pN type III collagen), but binding to triple helical collagen-like peptides is much weaker. Weak binding occurred to both hydroxylated and nonhydroxylated collagen-like peptides, and a significant chain length dependence was observed. Binding of HSP47 to native type I collagen had no effect on the thermal stability of the triple helix. Refolding of type I collagen in the presence of HSP47 showed minor changes, but these are probably not biologically significant. Binding of HSP47 to bovine pN type III collagen has only minor effects on the thermal stability of the triple helix and does not influence the refolding kinetics of the triple helix.     

Structure, dynamics, and thermodynamics of the structural domain of troponin C in complex with the regulatory peptide 1-40 of troponin I

The structure of the calcium-saturated C-domain of skeletal troponin C (CTnC) in complex with a regulatory peptide comprising residues 1-40 (Rp40) of troponin I (TnI) was determined using nuclear magnetic resonance (NMR) spectroscopy. The solution structure determined by NMR is similar to the structure of the C-domain from intact TnC in complex with TnI(1)(-)(47) determined by X-ray crystallography [Vassylyev, D. G., Takeda, S., Wakatsuki, S., Maeda, K., and Maeda, Y. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 4847-4852]. Changes in the dynamic properties of CTnC.2Ca2+ induced by Rp40 binding were investigated using backbone amide (15)N NMR relaxation measurements. Analysis of NMR relaxation data allows for extraction of motional order parameters on a per residue basis, from which the contribution of changes in picosecond to nanosecond time scale motions to the conformational entropy associated with complex formation can be estimated. The results indicate that binding of Rp40 decreases backbone flexibility in CTnC, particularly at the end of the C-terminal helix. The backbone conformational entropy change (-TDeltaS) associated with binding of Rp40 to CTnC.2Ca2+ determined from (15)N relaxation data is 9.6 +/- 0.7 kcal mol(-1) at 30 degrees C. However, estimation of thermodynamic quantities using a structural approach [Lavigne, P., Bagu, J. R., Boyko, R., Willard, L., Holmes, C. F., and Sykes, B. D. (2000) Protein Sci. 9, 252-264] reveals that the change in solvation entropy upon complex formation is dominant and overcomes the thermodynamic "cost" associated with "stiffening" of the protein backbone upon Rp40 binding. Additionally, backbone amide (15)N relaxation data measured at different concentrations of CTnC.2Ca2+.Rp40 reveal that the complex dimerizes in solution. Fitting of the apparent global rotational correlation time as a function of concentration to a monomer-dimer equilibrium yields a dimerization constant of approximately 8.3 mM.     

Stability junction at a common mutation site in the collagenous domain of the mannose binding lectin

Missense mutations in the collagen triple-helix that replace one of the required Gly residues in the (Gly-Xaa-Yaa)(n)() repeating sequence have been implicated in various disorders. Although most hereditary collagen disorders are rare, a common occurrence of a Gly replacement mutation is found in the collagenous domain of mannose binding lectin (MBL). A Gly --> Asp mutation at position 54 in MBL is found at a frequency as high as 30% in certain populations and leads to increased susceptibility to infections. The structural and energetic consequences of this mutation are investigated by comparing a triple-helical peptide containing the N-terminal Gly-X-Y units of MBL with the homologous peptide containing the Gly to Asp replacement. The mutation leads to a loss of triple-helix content but only a small decrease in the stability of the triple-helix (DeltaT(m) approximately 2 degrees C) and no change in the calorimetric enthalpy. NMR studies on specifically labeled residues indicate the portion of the peptide C-terminal to residue 54 is in a highly ordered triple-helix in both peptides, while residues N-terminal to the mutation site have a weak triple-helical signal in the parent peptide and are completely disordered in the mutant peptide. These results suggest that the N-terminal triplet residues are contributing little to the stability of this peptide, a hypothesis confirmed by the stability and enthalpy of shorter peptides containing only the region C-terminal to the mutation site. The Gly to Asp replacement at position 54 in MBL occurs at the boundary of a highly stable triple-helix region and a very unstable sequence. The junctional position of this mutation minimizes its destabilizing effect, in contrast with the significant destabilization seen for Gly replacements in peptides modeling collagen diseases.     

Molecular cloning of a novel ubiquitin-like protein, UBIN, that binds to ER targeting signal sequences

To identify proteins that interact with HSP47, an endoplasmic reticulum (ER)-resident molecular chaperone, a yeast two-hybrid screening was performed using mouse full-length HSP47 including an N-terminal signal sequence as a bait. Analysis of several positive clones led to the identification and cloning of a novel gene, ubin, encoding a ubiquitin-like protein. Unlike other ubiquitin-like proteins, UBIN was shown to interact with signal sequences of various secretory and ER-luminal proteins, including HSP47, but not interact with signal sequences of mitochondrial targeting in two-hybrid system. The possible function of UBIN will be discussed with regards to novel characteristics of binding to signal sequences for ER targeting.     

In vitro selection of state-specific peptide modulators of G protein signaling using mRNA display

The G protein regulatory (GPR) motif is a approximately 20-residue conserved domain that acts as a guanine dissociation inhibitor (GDI) for G(i/o)(alpha) subunits. Here, we describe the isolation of peptides derived from a GPR consensus sequence using mRNA display selection libraries. Biotinylated G(i)(alpha)(1), modified at either the N or C terminus, serves as a high-affinity binding target for mRNA-displayed GPR peptides. In vitro selection using mRNA display libraries based on the C terminus of the GPR motif revealed novel peptide sequences with conserved residues. Surprisingly, selected peptides contain mutations to a highly conserved Arg in the GPR motif, previously shown to be crucial for binding and inhibition activities. The dominant peptide from the selection, R6A, and a minimal 9-mer peptide, R6A-1, do not contain Arg residues yet retain high affinity (K(D) = 60 and 200 nM, respectively) and specificity for the GDP-bound state of G(i)(alpha)(1), as measured by surface plasmon resonance. The selected peptides also maintain GDI activity for G(i)(alpha)(1), inhibiting both the exchange of GDP in GTPgammaS binding assays and the AlF(4)(-)-stimulated enhancement of intrinsic tryptophan fluorescence. The kinetics of GDI activity, however, are different for the selected peptides and demonstrate biphasic kinetics, suggesting a complex mechanism for inhibition. Like the GPR motif, the R6A and R6A-1 peptides compete with G(betagamma) subunits for binding to G(i)(alpha)(1), suggesting their use as activators of G(betagamma) signaling.     

Cross-reactive binding of cyclic peptides to an anti-TGFalpha antibody Fab fragment: an X-ray structural and thermodynamic analysis.

The monoclonal antibody tAb2 binds the N-terminal sequence of transforming growth factor alpha, VVSHFND. With the help of combinatorial peptide libraries it is possible to find homologous peptides that bind tAb2 with an affinity similar to that of the epitope. The conformational flexibility of short peptides can be constrained by cyclization in order to improve their affinity to the antibody and their stability towards proteolysis. Two cyclic peptides which are cross-reactive binders for tAb2 were selected earlier using combinatorial peptide libraries. One is cyclized by an amide bond between the N-alpha group and the side-chain of the last residue (cyclo-SHFNEYE), and the other by a disulfide bridge (cyclo-CSHFNDYC). The complex structures of tAb2 with the linear epitope peptide VVSHFND and with cyclo-SHFNEYE were determined by X-ray diffraction. Both peptides show a similar conformation and binding pattern in the complex. The linear peptide SHFNEYE does not bind tAb2, but cyclo-SHFNEYE is stabilized in a loop conformation suitable for binding. Hence the cyclization counteracts the exchange of aspartate in the epitope sequence to glutamate. Isothermal titration calorimetry was used to characterize the binding energetics of tAb2 with the two cyclic peptides and the epitope peptide. The binding reactions are enthalpically driven with an unfavorable entropic contribution under all measured conditions. The association reactions are characterized by negative DeltaC(p) changes and by the uptake of one proton per binding site. A putative candidate for proton uptake during binding is the histidine residue in each of the peptides. Hydrogen bonds and the putative formation of an electrostatic pair between the protonated histidine and a carboxy group may contribute markedly to the favorable enthalpy of complex formation.Implications to cyclization of peptides for stabilization are discussed.     

Identification and characterization of peptides that bind to cyanovirin-N, a potent human immunodeficiency virus-inactivating protein

Cyanovirin-N (CV-N) exerts a potent human immunodeficiency virus (HIV)-inactivating activity against diverse strains of HIV by binding to the viral surface envelope glycoprotein gp120 and blocking its essential interactions with cellular receptors. Based on previous thermodynamic analyses, it has been speculated that discrete protein-protein interactions might play an important ancillary role in the CV-N/gp120 binding event, in addition to the interactions of CV-N with specific oligosaccharides present on gp120. Here, we report the identification and characterization of CV-N-binding peptides, which were isolated by screening of M13 phage-displayed peptide libraries. After performing three rounds of biopanning of the libraries against biotinylated CV-N, a CV-N-binding motif, X3CX6(W/F)(Y/F)CX2(Y/F), was evident. A vector was designed to express CV-N-binding peptides as a fusion with thioredoxin (Trx) containing a penta-His affinity tag. The CV-N-binding peptides fused with His-tagged Trx inhibited binding of the corresponding peptide-bearing phages to CV-N, confirming that the peptides possessed CV-N-binding activity. Optical biosensor binding studies showed that the one of the CV-N-binding peptide, TN10-1, bound to CV-N with a KD value of 1.9 microM. The results of alanine scanning mutagenesis of the peptide showed that aromatic residues at positions 11, 12, and 16, as well as the conformational structure of the peptide secured by a disulfide bond, were important for the binding interactions. A series of competitive binding assays confirmed that gp120 inhibited CV-N binding of the corresponding peptide-bearing phages, and suggested that TN10-1 peptides were mimicking the protein component of gp120 rather than mimicking specific oligosaccharides present on gp120.     

Correlation between symmetry breaker position and the preferences of conformationally constrained homopeptides: a molecular dynamics investigation

The conformational tendencies of C(alpha,alpha)-diethylglycine (Deg)-based peptides have been studied in solution using all atom molecular dynamics simulations. Specifically, the conformational effects of breaking the symmetry of the host Tfa-(Deg)(5)-OtBu (Tfa, trifluoroacetyl; OtBu, tert-butoxy) pentapeptide with punctual replacements at different sequence positions of one Deg residue by its corresponding guest chiral analogue, L-alpha-aminobutyric acid (L-Abu), have been examined by simulating the following peptides: Tfa-(Deg)(5)-OtBu, Tfa-(Deg)(2)-L-Abu-(Deg)(2)-OtBu, Tfa-(Deg)(3)-L-Abu-Deg-OtBu, and Tfa-(Deg)(4)-L-Abu-OtBu. Simulations show that only the Deg homopeptide is able to stabilize a 2.0(5) helix, even though a kinked arrangement with all the Deg residues adopting a fully-extended conformation was found to be stable when the L-Abu residue is introduced in the middle of the sequence. On the other hand, when the L-Abu residue is closer to the C-end of the sequence, the peptide chain prefers a partially folded 3(10)-helix. Additional simulations on Tfa-(Deg)(3)-L-Abu-(Deg)(3)-OtBu highlighted that, when the size of the Deg segments increases, their tendency to adopt a 2.0(5) helix predominates over the preferred folded conformation of L-Abu. The overall picture extracted after more than 300 ns of molecular dynamics simulation is that breaking the alpha-carbon symmetry of achiral C(alpha)-tetrasubstituted amino acids is a promising strategy to build up polypeptides with modulated conformational tendencies.     

Antithrombin III Basel. Identification of a Pro-Leu substitution in a hereditary abnormal antithrombin with impaired heparin cofactor activity

Antithrombin III Basel is a hereditary abnormal antithrombin with normal progressive inhibition activity (normal reactive site) and reduced heparin cofactor activity (impaired heparin binding site). Structures of antithrombin III Basel and normal antithrombin III isolated from the same patient were compared by peptide mapping using the dimethylaminoazobenzene isothiocyanate precolumn derivatization technique. Of the approximately 50 tryptic peptides of normal and abnormal antithrombin III, one peptide comprising residues 40-46 had a different retention time in reversed-phase high performance liquid chromatography. The amino acid sequence of the peptide from antithrombin III Basel had a single substitution of Pro (normal) by Leu (abnormal) at position 41. This substitution is close to an Arg (residue 47) and a Trp (residue 49) which have previously been shown to be critical for heparin binding by antithrombin III. Although additional amino acid substitutions in antithrombin III Basel cannot be ruled out, this Pro-Leu replacement could cause a conformational change by increasing both the helical structure and the hydrophobicity around residue 41. These data suggest that: (i) the heparin binding site of antithrombin III encompasses the region containing residues 41, 47, and 49; and (ii) the impaired heparin cofactor activity of antithrombin III Basel is likely due to a conformational change of the heparin binding site induced by the Pro-Leu substitution at position 41.     

Discovery of substrate for type I signal peptidase SpsB from Staphylococcus aureus.

Based on the kinetic model of substrate phage proteolysis, we have formulated a strategy for best manipulating the conditions in screening phage display libraries for protease substrates (Sharkov, N. A., Davis, R. M., Reidhaar-Olson, J. F., Navre, M., and Cai, D. (2001) J. Biol. Chem. 276, 10788-10793). This strategy is exploited in the present study with signal peptidase SpsB from Staphylococcus aureus. We demonstrate that highly active substrate phage clones can be isolated from a phage display library by systematically tuning the selection stringency in screening. Several of the selected clones exhibit superior reactivity over a control, the best clone, SIIIRIII-8, showing >100-fold improvement. Because no conserved sequence features were readily revealed that could allow delineation of the active and unreactive clones, the sequences identified in five of the active clones were tested as synthetic dodecamers, Ac-AGX(8)GA-NH(2). Using electrospray ionization mass spectrometry, we show that four of these peptides can be cleaved by SpsB and that Ala is the P1 residue exclusively and Ala or Leu the P3 residue, in keeping with the (-3, -1) rule for substrate recognition by signal peptidase. Our successful screening with SpsB demonstrated the general applicability of the screening strategy and allowed us to isolate the first peptide substrates for the enzyme.