共查询到20条相似文献,搜索用时 19 毫秒
1.
Mohd Akif Aman Mahajan Edward D. Sturrock K. Ravi Acharya 《Journal of molecular biology》2010,400(3):502-3307
Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE. 相似文献
2.
Fernandez JH Hayashi MA Camargo AC Neshich G 《Biochemical and biophysical research communications》2003,308(2):219-226
Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase which converts angiotensin I into the vasopressor peptide angiotensin II and also inactivates the hypotensive peptide bradykinin, playing an important role in blood pressure regulation. The present work describes the molecular modeling of the N-terminal human somatic ACE in complex with the inhibitor lisinopril, identifying the residues involved in the inhibitor-binding pocket. The obtained results identify differences in the lisinopril lysine moiety-binding residues for N- and C-terminals of sACE domains and an important carboxy-terminal proline hydrophobic accommodations mediated by the aromatic ring of Tyr532 and Tyr1128 residues, respectively. The present model will be useful for the development of a new inhibitor family based on the natural BPP peptides and derivatives, or even to improve the binding capacities and the domain specificity of the already known inhibitors. 相似文献
3.
Yamamoto D Takai S Miyazaki M 《Biochemical and biophysical research communications》2007,354(4):981-984
To characterize the inhibitory specificity of angiotensin converting enzyme (ACE) inhibitors for matrix metalloproteinase 9 (MMP-9) activity, molecular modeling of these complex was performed referring the recent X-ray structure analyses using lisinopril as an ACE inhibitor. Two interaction modes differing in the orientation of the inhibitor on the active site were identified. Lisinopril was effectively stabilized by specific hydrogen bonds and hydrophobic interactions in the active site of MMP-9, and its hydrophobic group appeared to interact preferentially with the S1 site compared with the S1' site. These findings showed that ACE inhibitors could become important seeds for cardiovascular protection and the development of MMP inhibitors. 相似文献
4.
Corradi HR Schwager SL Nchinda AT Sturrock ED Acharya KR 《Journal of molecular biology》2006,357(3):964-974
Human somatic angiotensin I-converting enzyme (sACE) is a key regulator of blood pressure and an important drug target for combating cardiovascular and renal disease. sACE comprises two homologous metallopeptidase domains, N and C, joined by an inter-domain linker. Both domains are capable of cleaving the two hemoregulatory peptides angiotensin I and bradykinin, but differ in their affinities for a range of other substrates and inhibitors. Previously we determined the structure of testis ACE (C domain); here we present the crystal structure of the N domain of sACE (both in the presence and absence of the antihypertensive drug lisinopril) in order to aid the understanding of how these two domains differ in specificity and function. In addition, the structure of most of the inter-domain linker allows us to propose relative domain positions for sACE that may contribute to the domain cooperativity. The structure now provides a platform for the design of "domain-specific" second-generation ACE inhibitors. 相似文献
5.
Hypertension (high blood pressure) is a major risk factor for cardiovascular disease, which is the leading cause of death worldwide. The somatic isoform of angiotensin I‐converting enzyme (sACE) plays a critical role in blood pressure regulation, and ACE inhibitors are thus widely used to treat hypertension and cardiovascular disease. Our current understanding of sACE structure, dynamics, function, and inhibition has been limited because truncated, minimally glycosylated forms of sACE are typically used for X‐ray crystallography and molecular dynamics simulations. Here, we report the first cryo‐EM structures of full‐length, glycosylated, soluble sACE (sACES1211). Both monomeric and dimeric forms of the highly flexible apo enzyme were reconstructed from a single dataset. The N‐ and C‐terminal domains of monomeric sACES1211 were resolved at 3.7 and 4.1 Å, respectively, while the interacting N‐terminal domains responsible for dimer formation were resolved at 3.8 Å. Mechanisms are proposed for intradomain hinging, cooperativity, and homodimerization. Furthermore, the observation that both domains were in the open conformation has implications for the design of sACE modulators. 相似文献
6.
RXPA380 (Cbz-PhePsi[PO(2)CH]Pro-Trp-OH) was reported recently as the first highly selective inhibitor of the C-domain of somatic angiotensin-converting enzyme (ACE), able to differentiate the two active sites of somatic ACE by a selectivity factor of more than 3 orders of magnitude. The contribution of each RXPA380 residue toward this remarkable selectivity was evaluated by studying several analogues of RXPA380. This analysis revealed that both pseudo-proline and tryptophan residues in the P(1)' and P(2)' positions of RXPA380 play a critical role in the selectivity of this inhibitor for the C-domain. This selectivity is not due to a preference of the C-domain for inhibitors bearing pseudo-proline and tryptophan residues, but rather reflects the poor accommodation of these inhibitor residues by the N-domain. A model of RXPA380 in complex with the ACE C-domain, based on the crystal structure of germinal ACE, highlights residues that may contribute to RXPA380 selectivity. From this model, striking differences between the N- and C-domains of ACE are observed for residues defining the S(2)' pocket. Of the twelve residues that surround the tryptophan side chain of RXPA380 in the C-domain, five are different in the N-domain. These differences in the S(2)' composition between the N- and C-domains are suggested to contribute to RXPA380 selectivity. The structural insights provided by this study should enhance understanding of the factors controlling the selectivity of the two domains of somatic ACE and allow the design of new selective ACE inhibitors. 相似文献
7.
The structure of testis angiotensin-converting enzyme in complex with the C domain-specific inhibitor RXPA380 总被引:1,自引:0,他引:1
Corradi HR Chitapi I Sewell BT Georgiadis D Dive V Sturrock ED Acharya KR 《Biochemistry》2007,46(18):5473-5478
Angiotensin I-converting enzyme (ACE) is central to the regulation of the renin-angiotensin system and is a key therapeutic target for combating hypertension and related cardiovascular diseases. Currently available drugs bind both active sites of its two homologous domains, although it is now understood that these domains function differently in vivo. The recently solved crystal structures of both domains (N and C) open the door to new domain-specific inhibitor design, taking advantage of the differences between these two large active sites. Here we present the first crystal structure at a resolution of 2.25 A of testis ACE (identical to the C domain of somatic ACE) with the highly C-domain-specific phosphinic inhibitor, RXPA380. Testis ACE retains the same conformation as seen in previously determined inhibitor complexes, but the RXPA380 central backbone conformation is more similar to that seen for the inhibitor captopril than enalaprilat. The RXPA380 molecule occupies more subsites of the testis ACE active site than the previously determined inhibitors and possesses bulky moieties that extend into the S2' and S2 subsites. Thus the high affinity of RXPA380 for the testis ACE/somatic ACE C domain is explained by the interaction of these bulky moieties with residues unique to these domains, specifically Phe 391, Val 379, and Val 380, that are not found in the N domain. The characterization of the extended active site and the binding of a potent C-domain-selective inhibitor provide the first structural data for the design of truly domain-specific pharmacophores. 相似文献
8.
Akif M Schwager SL Anthony CS Czarny B Beau F Dive V Sturrock ED Acharya KR 《The Biochemical journal》2011,436(1):53-59
Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97? (1 ?=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15?) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P(1)' residue with the unusual R configuration which, surprisingly, is accommodated by the large S(2)' pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi-pi stacking interactions with the amino benzoyl group of the first molecule locking them in a 'hand-shake' conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors. 相似文献
9.
The interaction of P1 and P3 side chains with the combining S1 and S3 hydrophobic subsites of HIV and FIV proteases has been explored using asymmetric competitive inhibitors. The inhibitors evaluated contained (2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acid (allophenylnorstatine) as the hydroxymethylcarbonyl isostere, (R)-5,5-dimethyl-1, 3-thiazolidine-4-carbonyl as P1', Val as P2 and P2' residues, and a variety of amino acids at the P3 and P3' positions. All inhibitors showed competitive inhibition of both enzymes with higher potency against the HIV protease in vitro. Within this series, 31 (VLE776) is the most effective inhibitor against FIV protease, and it contains Phe at P3, but no P3' residue. VLE776 also exhibited potent antiviral activities against the drug-resistant HIV mutants (G48V and V82F) and the TL3-resistant HIV mutants. Explanation of the inhibition activities was described. In addition, a new strategy was described for development of bifunctional inhibitors, which combine the protease inhibitor and another enzyme inhibitor in one molecule. 相似文献
10.
Human angiotensin-converting enzyme (ACE) has two homologous domains, the N and C domains, with differing substrate preferences. X-ray crystal structures of the C and N domains complexed with various inhibitors have allowed identification of active site residues that might be important for the molecular basis of this selectivity. However, it is unclear to what extent the different residues contribute to substrate domain selectivity. Here, cocrystal structures of human testis ACE, equivalent to the C domain, have been determined with two novel C domain-selective ketomethylene inhibitors, (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-tryptophan (kAW) and (5 S)-5-[( N-benzoyl)amino]-4-oxo-6-phenylhexanoyl- l-phenylalanine (kAF). The ketone groups of both inhibitors bind to the zinc ion as a hydrated geminal diolate, demonstrating the ability of the active site to catalyze the formation of the transition state. Moreover, active site residues involved in inhibitor binding have been mutated to their N domain counterparts, and the effect of the mutations on inhibitor binding has been determined. The C domain selectivity of these inhibitors was found to result from interactions between bulky hydrophobic side chain moieties and C domain-specific residues F391, V518, E376, and V380 (numbering of testis ACE). Mutation of these residues decreased the affinity for the inhibitors 4-20-fold. T282, V379, E403, D453, and S516 did not contribute individually to C domain-selective inhibitor binding. Further domain-selective inhibitor design should focus on increasing both the affinity and selectivity of the side chain moieties. 相似文献
11.
Novel peptidomimics as angiotensin-converting enzyme inhibitors: a combinatorial approach 总被引:3,自引:0,他引:3
One of the efficient mode of treatments of chronic hypertension and cardiovascular disorders has been to restrain the formation of angiotensin-II by inhibiting the action of angiotensin-converting enzyme (ACE) on angiotensin-I. A number of ACE inhibitors (ACEIs) have been put to therapeutic use during the last two decades. The efforts continue towards achieving superior molecules or drugs with improved affinities, better bioavailability and thus long duration of action with minimum side effects. The present work evolves around similar objectives. In order to understand the mode of interaction of inhibitors with the active site of the enzyme and subsequently to have lead compounds as possible inhibitors the novel dipeptidomimics and tripeptidomimics have been designed and synthesized using combinatorial chemistry approach. A Focussed library of 10 di- and tri-peptides, eight dipeptidomemics and forty tripeptidomemics was generated. The pharmacophoric heterocyclic moieties and the amino acids have been selected to have affinities with the S1, S1', and S2' subsites of the active site of the enzyme. ACE inhibition studies clearly demonstrated the structural-activity relationships within these classes of peptidomimics. The dipeptidomimics interacted only with S1' and S2' subsites, whereas the tripeptidomemics had additional interaction with S1 subsite, which accounted for their significant ACE inhibition potencies. The in-vitro screening of these peptidomimics have resulted in identification of four promising tripeptidomimics 34[2-benzimidazolepropionyl-Val-Trp], 35[5hydroxytryptophanyl-Val-Trp], 40[2-benzimidazolepropionyl-Ile-Trp] and 45[2-benzimidazolepropionyl-Lys-Trp] with IC50 values in micromolar concentrations. 相似文献
12.
T Benchetrit M C Fournié-Zaluski B P Roques 《Biochemical and biophysical research communications》1987,147(3):1034-1040
The inhibitory potency of separate enantiomers of thiorphan and retrothiorphan has shown that several particularities of the active site of thermolysin are also present in the neutral endopeptidase 24.11, "enkephalinase", such as its ability: i) to recognize a retroamide bond as well as a standard amide bond, ii) to interact similarly with residues in P1' position of either R or S configuration in the thiorphan series but contrastingly to discriminate between the R and S isomers in the retrothiorphan series. These four inhibitors were modellized in the thermolysin active site and their spatial arrangement compared with that of a thiol inhibitor co-crystallized with thermolysin. In all cases, the essential interactions involved in the stabilization of the bound inhibitor were conserved. However, the bound (R) retrothiorphan displayed unfavorable intramolecular contacts, accounting for its lower inhibitory potency for the two metallopeptidases. 相似文献
13.
Coates D Isaac RE Cotton J Siviter R Williams TA Shirras A Corvol P Dive V 《Biochemistry》2000,39(30):8963-8969
Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two homologous protein domains, resulting from a tandem gene duplication. It has been proposed that the N- and C-terminal active sites can have specific in vivo roles. In Drosophila melanogaster, Ance and Acercode for two ACE-like single-domain proteins, also predicted to have distinct physiological roles. We have investigated the relationship of Ance and Acer to the N- and C-domains of human sACE by genomic sequence analysis and by using domain-selective inhibitors, including RXP 407, a selective inhibitor of the human N-domain. These phosphinic peptides were potent inhibitors of Acer, but not of Ance. We conclude that the active sites of the N-domain and of Acer share structural features that permit the binding of the unusual RXP407 inhibitor and the hydrolysis of a broader range of peptide structures. In comparison, Ance, like the human C-domain of ACE, displays greater inhibitor selectivity. From the analysis of the published sequence of the Adh region of Drosophila chromosome 2, which carries Ance, Acer, and four additional ACE-like genes, we also suggest that this functional conservation is reflected in an ancestral gene structure identifiable in both protostome and deuterostome lineages and that the duplication seen in vertebrate genomes predates the divergence of these lineages. The conservation of ACE enzymes with distinct active sites in the evolution of both vertebrate and invertebrate species provides further evidence that these two kinds of active sites have different physiological functions. 相似文献
14.
Angiotensin-converting enzyme (ACE), a membrane-bound zinc metallopeptidase, catalyzes the formation of Angiotensin-II (AngII) and the deactivation of bradykinin in the renin–angiotensin-aldosterone and kallikrein–kinin systems. As a hydrolysis product of ACE, AngII is regarded as an inhibitor and displays stronger competitive inhibition in the C-domain than the N-domain of ACE. However, the AngII binding differences between the two domains and the mechanisms behind AngII dissociation from the C-domain are rarely explored. In this work, molecular docking, Molecular Mechanics/Poisson–Boltzmann Surface Area calculation, and steered molecular dynamics (SMD) are applied to explore the structures and interactions in the binding or unbinding of AngII with the two domains of human somatic ACE. Calculated free energy values suggest that the C-domain–AngII complex is more stable than the N-domain–AngII complex, consistent with available experimental data. SMD simulation results imply that electrostatic interaction is dominant in the dissociation of AngII from the C-domain. Moreover, Gln106, Asp121, Glu123, and Tyr213 may be the key residues in the unbinding pathway of AngII. The simulation results in our work provide insights into the interactions between the two domains of ACE and its natural peptide inhibitor AngII at a molecular level. Moreover, the results provide theoretical clues for the design of new inhibitors. 相似文献
15.
Deaton DN Graham KP Gross JW Miller AB 《Bioorganic & medicinal chemistry letters》2008,18(5):1681-1687
Explorations of the S(1') subsite of ACE2 via modifications of the P(1') methylene biphenyl moiety of thiol-based metalloprotease inhibitors led to improvements in ACE2 selectivity versus ACE and NEP, while maintaining potent ACE2 inhibition. 相似文献
16.
El-Kabbani O Rogniaux H Barth P Chung RP Fletcher EV Van Dorsselaer A Podjarny A 《Proteins》2000,41(3):407-414
Aldose and aldehyde reductases are monomeric NADPH-dependent oxidoreductases that catalyze the reduction of a wide variety of aldehydes and ketones to their corresponding alcohols. The overall three-dimensional structures of the enzymes are composed of similar alpha/beta TIM-barrels, and the active site residues Tyr 50, His 113, and Trp 114 interacting with the hydrophilic heads of inhibitors are conserved. We have used molecular modeling and mass spectrometry to characterize the interactions between the enzymes and three aldose reductase inhibitors: tolrestat, sorbinil, and zopolrestat. Unlike the IC(50) values (concentration of inhibitor giving 50% of inhibition in solution), the Vc(50) values measured by mass spectrometry (accelerating voltage of ions needed to dissociate 50% of a noncovalent complex in the gas phase) for the two enzymes are similar, and they correlate with the electrostatic and hydrogen-bonding energies calculated between the conserved Tyr 50, His 113, and Trp 114 and the inhibitors. The results of our comparison agree with detailed structural information obtained by X-ray crystallography, suggesting that nonconserved residues from the C-terminal loop account for differences in IC(50) values for the two enzymes. Additionally, they confirm our previous assumption that the Vc(50) values reflect the enzyme-inhibitor electrostatic and hydrogen-bonding interactions and exclude the hydrophobic interactions. 相似文献
17.
Mahalingam B Boross P Wang YF Louis JM Fischer CC Tozser J Harrison RW Weber IT 《Proteins》2002,48(1):107-116
HIV-1 develops resistance to protease inhibitors predominantly by selecting mutations in the protease gene. Studies of resistant mutants of HIV-1 protease with single amino acid substitutions have shown a range of independent effects on specificity, inhibition, and stability. Four double mutants, K45I/L90M, K45I/V82S, D30N/V82S, and N88D/L90M were selected for analysis on the basis of observations of increased or decreased stability or enzymatic activity for the respective single mutants. The double mutants were assayed for catalysis, inhibition, and stability. Crystal structures were analyzed for the double mutants at resolutions of 2.2-1.2 A to determine the associated molecular changes. Sequence-dependent changes in protease-inhibitor interactions were observed in the crystal structures. Mutations D30N, K45I, and V82S showed altered interactions with inhibitor residues at P2/P2', P3/P3'/P4/P4', and P1/P1', respectively. One of the conformations of Met90 in K45I/L90M has an unfavorably close contact with the carbonyl oxygen of Asp25, as observed previously in the L90M single mutant. The observed catalytic efficiency and inhibition for the double mutants depended on the specific substrate or inhibitor. In particular, large variation in cleavage of p6(pol)-PR substrate was observed, which is likely to result in defects in the maturation of the protease from the Gag-Pol precursor and hence viral replication. Three of the double mutants showed values for stability that were intermediate between the values observed for the respective single mutants. D30N/V82S mutant showed lower stability than either of the two individual mutations, which is possibly due to concerted changes in the central P2-P2' and S2-S2' sites. The complex effects of combining mutations are discussed. 相似文献
18.
Papakyriakou A Spyroulias GA Sturrock ED Manessi-Zoupa E Cordopatis P 《Biochemistry》2007,46(30):8753-8765
Human angiotensin-I converting enzyme (ACE) is a central component of the renin-angiotensin system and a major target for cardiovascular therapies. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. On the basis of the recently determined crystal structures of both ACE domains, we have studied their complexes with gonadotropin-releasing hormone (GnRH), which is cleaved releasing both the protected NH2- and COOH-terminal tripeptides. This is the first molecular modeling study of an ACE-peptide substrate complex that examines the structural basis of ACE's endopeptidase activity and offers novel insights into subsites that are distant from the obligatory binding site and were not identified in the crystal structures. Our data indicate that a bridging interaction between Arg500 of the N-domain and Arg8 of GnRH that involves a buried chloride ion may account for its role in the specificity of the N-domain for endoproteolytic cleavage of the substrate at the NH2-terminus in vitro. In support of this, the protected NH2-terminal dipeptide of GnRH exhibits stronger interactions than the protected COOH-terminal dipeptide with the N-domain of ACE. Further comparison of the models of ACE-substrate complexes promotes our understanding of how the two domains differ in their function and specificity and provides an extension of the pharmacophore model used for structure-based drug design up to the S7 subsite of the enzyme. 相似文献
19.
Apoptosis, or programmed cell death, plays a central role in the development and homeostasis of an organism. The breakdown of cellular proteins in apoptosis is mediated by caspases, which comprise a highly conserved family of cysteine proteases with specificity for aspartic acid residues at the P1 positions of their substrates. Multiple lines of evidence show that caspase-9 is critical for an apoptosis pathway mediated via the mitochondria. In this study, the three-dimensional structure of the catalytic domain of caspase-9 and its interaction with the inhibitor acetyl-Asp-Val-Ala-Asp fluoromethyl ketone (Ac-DVAD-fmk) have been predicted by a segment matching modeling procedure. As expected, the predicted caspase-9 structure shows both a high similarity in the overall folding topology and remarkable differences in the surface loop regions as compared to other caspase family members such as caspase-1, -3 and -8, for which crystal structures have been determined. This kind of comparative analysis reflects the convergence-divergence duality among the caspases. Moreover, some subtle differences have been observed between caspase-9 and caspase-3 in the subsite contacts with the covalently linked inhibitor Ac-DVAD-fmk. Based on the X-ray structural analysis of caspase-8, a main chain carbonyl oxygen appears to be involved in a catalytic triad with the active site Cys and His residues. The corresponding carbonyl oxygen in caspase-9, together with other expected features of the catalytic apparatus, appears in our model. The predicted structure of caspase-9 can serve as a reference for subsite analysis relative to rational design of highly selective caspase inhibitors for therapeutic application. 相似文献
20.
J. Liu Y. Gong O. Prakash L. Wen I. Lee J. K. Huang R. Krishnamoorthi 《Protein science : a publication of the Protein Society》1998,7(1):132-141
Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49:593-626), whereby an enzyme-catalyzed equilibrium between intact (I) and reactive-site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd, is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI-III; Mr 3 kDa) of the squash family and rCMTI-V (Mr approximately 7 kDa) of the potato I family. These two inhibitors have different binding loop-scaffold interactions and different Khyd values--2.4 (CMTI-III) and 9 (CMTI-V)--at 25 degrees C. The reactive-site peptide bond (P1-P1') is that between Arg5 and Ile6 in CMTI-III, and that between Lys44 and Asp45 in CMTI-V. The order parameters (S2) of backbone NHs of uniformly 15N-labeled rCMTI-III and rCMTI-III* were determined from measurements of 15N spin-lattice and spin-spin relaxation rates, and [1H]-15N steady-state heteronuclear Overhauser effects, using the model-free formalism, and compared with the data reported previously for rCMTI-V and rCMTI-V*. The backbones of rCMTI-III [(S2) = 0.71] and rCMTI-III* [(S2) = 0.63] are more flexible than those of rCMTI-V [(S2) = 0.83] and rCMTI-V* [(S2) = 0.85]. The binding loop residues, P4-P1, in the two proteins show the following average order parameters: 0.57 (rCMTI-III) and 0.44 (rCMTI-III*); 0.70 (rCMTI-V) and 0.40 (rCMTI-V*). The P1'-P4' residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI-III) and 0.47 (rCMTI-III*); and 0.73 (rCMTI-V) and 0.83 (rCMTI-V*). The newly formed C-terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI-III* due to the Cys3-Cys20 crosslink. In contrast, the newly formed N-terminal (Pn' residues) becomes more flexible only in rCMTI-III*, most likely due to lack of an interaction between the P1' residue and the scaffold in rCMTI-III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn' residues seem to facilitate the resynthesis of the P1-P1' bond, leading to a lower Khyd value. 相似文献