Intramolecular Bridges Research Articles

Bis(5-tert-butyl-2-hydroxy-3-methylbenzyl)(6-hydroxyhexyl)ammonium chloride monohydrate, an anion receptor complex

In the title compound, C(30)H(48)NO(3)(+) x Cl(-) x H(2)O, the cation acts with a water molecule as a chloride ion receptor. The chloride ion forms three strong intramolecular hydrogen bonds. The water molecule forms both an intramolecular bridge between one phenol H atom and the chloride ion, and an intermolecular link to the aliphatic alcohol O atom. Weak intermolecular C-H...Cl and C-H ...O hydrogen bonds provide additional packing interactions.

Redox-mediated substrate recognition by Sdp1 defines a new group of tyrosine phosphatases

Reactive oxygen species trigger cellular responses by activation of stress-responsive mitogen-activated protein kinase (MAPK) signalling pathways. Reversal of MAPK activation requires the transcriptional induction of specialized cysteine-based phosphatases that mediate MAPK dephosphorylation. Paradoxically, oxidative stresses generally inactivate cysteine-based phosphatases by thiol modification and thus could lead to sustained or uncontrolled MAPK activation. Here we describe how the stress-inducible MAPK phosphatase, Sdp1, presents an unusual solution to this apparent paradox by acquiring enhanced catalytic activity under oxidative conditions. Structural and biochemical evidence reveals that Sdp1 employs an intramolecular disulphide bridge and an invariant histidine side chain to selectively recognize a tyrosine-phosphorylated MAPK substrate. Optimal activity critically requires the disulphide bridge, and thus, to the best of our knowledge, Sdp1 is the first example of a cysteine-dependent phosphatase that couples oxidative stress with substrate recognition. We show that Sdp1, and its paralogue Msg5, have similar properties and belong to a new group of phosphatases unique to yeast and fungal taxa.

A Novel Lantibiotic Acting on Bacterial Cell Wall Synthesis Produced by the Uncommon Actinomycete Planomonospora sp.

Important classes of antibiotics acting on bacterial cell wall biosynthesis, such as beta-lactams and glycopeptides, are used extensively in therapy and are now faced with a challenge because of the progressive spread of resistant pathogens. A discovery program was devised to target novel peptidoglycan biosynthesis inhibitors capable of overcoming these resistance mechanisms. The microbial products were first screened according to their differential activity against Staphylococcus aureus and its L-form. Then, activities insensitive to the addition of a beta-lactamase cocktail or d-alanyl-d-alanine affinity resin were selected. Thirty-five lantibiotics were identified from a library of broth extracts produced by 40,000 uncommon actinomycetes. Five of them showed structural characteristics that did not match with any known microbial metabolite. In this study, we report on the production, structure determination, and biological activity of one of these novel lantibiotics, namely, planosporicin, which is produced by the uncommon actinomycete Planomonospora sp. Planosporicin is a 2194 Da polypeptide originating from 24 proteinogenic amino acids. It contains lanthionine and methyllanthionine amino acids generating five intramolecular thioether bridges. Planosporicin selectively blocks peptidoglycan biosynthesis and causes accumulation of UDP-linked peptidoglycan precursors in growing bacterial cells. On the basis of its mode of action and globular structure, planosporicin can be assigned to the mersacidin (20 amino acids, 1825 Da) and the actagardine (19 amino acids, 1890 Da) subgroup of type B lantibiotics. Considering its spectrum of activity against Gram-positive pathogens of medical importance, including multi-resistant clinical isolates, and its efficacy in vivo, planosporicin represents a potentially new antibiotic to treat emerging pathogens.

Investigation of the complexation of the anti-cancer drug novantrone with the hairpin structure of the deoxyheptanucleotide 5′-d(GpCpGpApApGpC)

In aqueous solution the deoxyheptanucleotide, 5′-d(GpCpGpApApGpC), exists as a very stable hairpin structure in equilibrium with small proportions of the single-stranded and duplex forms. Complexation of the anti-cancer drug novantrone (mitoxantrone) with the DNA heptamer was investigated by one- and two-dimensional 500 MHz 1H NMR spectroscopy (2M-TOCSY, 2M-NOESY) and molecular dynamics simulations. The proton chemical shifts of NOV in mixed solutions with the heptamer were measured as a function of concentration and temperature and the equilibrium association parameters were determined for complexation of NOV with the three forms of the heptamer. The spatial structure of the complex of the antibiotic with the hairpin form of the heptamer was built on the basis of 2D-NOE data. The conformational dynamics of the complex and its interaction with the water environment were investigated by molecular dynamics methods. The results suggest that NOV complexes with the hairpin form of the heptamer in solution by intercalation. Complexation of NOV with the hairpin stem results in a disruption of about one half of the intramolecular water bridges of the hairpin, which is considered to be the main reason for the observed decrease in the thermodynamical stability of the hairpin on binding with the ligand.

E/Z Isomerization of 3-Hydrazonocamphor Promoted by Coordination to Palladium or Platinum

3-Hydrazonocamphor, 3-(RR1NN)C10H14O (R = Me, R1 = H), undergoes intramolecular hydrogen bridging by coordination to platinum or palladium. This effect is evidenced by considerable decrease in the ν(C=O) frequency (compared to the free ligand) in the IR spectra of the complexes [MCl2L2] (M = Pd, Pt; L = 3-(RR1NN)C10H14O) as well as by the magnetic non- equivalence of the two ligands, as revealed by 13C NMR. DFT calculations indicate that coordination of 3-(Me(H)NN)C10H14O promotes E/Z isomerization of the hydrazono group of the ligand, inducing formation of intramolecular hydrogen bonding and corresponding stabilization of the complex. Characterization of the complexes [MCl2L2] (M = Pt; L: R, R1 = Me (1), R = Me, R1 = H (2) and M = Pd; L: R = Me, R1 = H (3)) was performed by analytical and spectroscopic techniques. Redox properties of the 3-hydazonocamphors and their complexes were studied by cyclic voltammetry. The structure of trans-[PtCl2{3-(Me2NN)C10H14O}2] was determined by single-crystal X-ray diffraction analysis. The complex has square-planar geometry and crystallizes in the tetragonal P43 space group.

A new dinuclear heme-copper complex derived from functionalized protoporphyrin IX

A new biomimetic model for the heterodinuclear heme/copper center of respiratory oxidases is described. It is derived from iron(III) protoporphyrin IX by covalent attachment of a Gly-L-His-OMe residue to one propionic acid substituent and an amino-bis(benzimidazole) residue to the other propionic acid substituent of the porphyrin ring, yielding the Fe(III) complex 1, and subsequent addition of a copper(II) or copper(I) ion, according to needs. The fully oxidized Fe(III)/Cu(II) complex, 2, binds azide more strongly than 1, and likely contains azide bound as a bridging ligand between Fe(III) and Cu(II). The two metal centers also cooperate in the reaction with hydrogen peroxide, as the peroxide adducts obtained at low temperature for 1 and 2 display different optical features. Support to this interpretation comes from the investigation of the peroxidase activity of the complexes, where the activation of hydrogen peroxide has been studied through the phenol coupling reaction of p-cresol. Here the presence of Cu(II) improves the catalytic performance of complex 2 with respect to 1 at acidic pH, where the positive charge of the Cu(II) ion is useful to promote O-O bond cleavage of the iron-bound hydroperoxide, but it depresses the activity at basic pH because it can stabilize an intramolecular hydroxo bridge between Fe(III) and Cu(II). The reactivity to dioxygen of the reduced complexes has been studied at low temperature starting from the carbonyl adducts of the Fe(II) complex, 3, and Fe(II)/Cu(I) complex, 4. Also in this case the adducts derived from the Fe(II) and Fe(II)/Cu(I) complexes, that we formulate as Fe(III)-superoxo and Fe(III)/Cu(II)-peroxo exhibit slightly different spectral properties, showing that the copper center participates in a weak interaction with the dioxygen moiety.

Synthesis and Transition Metal Complexes of Novel N,N,O Scorpionate Ligands Suitable for Solid Phase Immobilisation

AbstractIntroduction of an allyl or a hydroxymethyl group to bis(3,5‐dimethylpyrazol‐1‐yl)acetic acid (1) at the bridging carbon atom leads to the new tripodal N,N,O ligands 2,2‐bis(3,5‐dimethylpyrazol‐1‐yl)pent‐4‐enoic acid (Hbdmpzpen) (2) and 2,2‐bis(3,5‐dimethylpyrazol‐1‐yl)‐3‐hydroxypropionic acid (Hbdmpzhp) (3). These ligands exhibit similar chemical behaviour to that of 1, but they have the additional possibility to be immobilised to a solid phase. Esterification of the hydroxymethyl linker of 3 yields 2,2‐bis(3,5‐dimethylpyrazol‐1‐yl)‐3‐acetatopropionic acid (Hbdmpzap) (4). The molecular structures of 2, 3 and 4 all exhibit intramolecular hydrogen bridges. Introduction of a hydroxymethyl group to methyl bis(3,5‐dimethylpyrazol‐1‐yl)acetate (5) yields methyl2,2‐bis(3,5‐dimethylpyrazol‐1‐yl)‐3‐hydroxypropionate(Mebdmpzhp) (6), which can be immobilised on Merrifield polymer to yield modified resin P‐6. To investigate the reactivity of these new ligands, manganese and rhenium complexes of 2, 3 and 4 have been studied. The molecular structures of the two manganese complexes [Mn(bdmpzpen)(CO)3] (7) and [Mn(bdmpzap)(CO)3] (8) have been confirmed by single‐crystal X‐ray structure determination. Saponification of polymer resin P‐6 and subsequent reaction with [ReBr(CO)5] yields rhenium tricarbonyl complexes anchored on Merrifield polymer (P‐Re). Solid phase immobilisation of the [Mn(bdmpzpen)(CO)3] (7) and [Re(bdmpzpen)(CO)3] (9) complexes on 3‐mercaptopropyl functionalised silica is initialised by AIBN. The tripodal coordination of manganese and rhenium in these functionalised Merrifield resins (P‐Re) and silica (S‐Mn, S‐Re) is proven by a single A1 and two E signals in the IR spectra that are typical for unsymmetrical “piano stool” type carbonyl complexes. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2006)

Dynamics of Asp23−Lys28 Salt-Bridge Formation in Aβ10-35Monomers

In the amyloid fibrils formed from long fragments of the amyloid beta-protein (Abeta-protein), the monomers are arranged in parallel and lie perpendicular to the fibril axis. The structure of the monomers satisfies the amyloid self-organization principle; namely, the low free energy state of the monomer maximizes the number of intra- and interpeptide contacts and salt bridges. The formation of the intramolecular salt bridge between Asp(D)23 and Lys(K)28 ensures that unpaired charges are not buried in the low-dielectric interior. We have investigated, using all-atom molecular dynamics simulations in explicit water, whether the D23-K28 interaction forms spontaneously in the isolated Abeta10-35 monomer. To validate the simulation protocol, we show, using five independent trajectories spanning a total of 100 ns, that the pKa values of the titratable groups are in good agreement with experimental measurements. The computed free energy disconnectvity graph shows that broadly the ensemble of compact random coil conformations can be clustered into four basins that are separated by free energy barriers ranging from 0.3 to 2.7 kcal/mol. There is significant residual structure in the conformation of the peptide in each of the basins. Due to the desolvation penalty, the structural motif with a stable turn involving the residues VGSN and a preformed D23-K28 contact is a minor component of the simulated structures. The extent of solvation of the peptides in the four basins varies greatly, which underscores the dynamical fluctuations in the monomer. Our results suggest that the early event in the oligomerization process must be the expulsion of discrete water molecules that facilitates the formation of interpeptide-interaction-driven stable structures with an intramolecular D23-K28 salt bridge and an intact VGSN turn.

HPLC–MS characterization of cyclo‐statherin Q‐37, a specific cyclization product of human salivary statherin generated by transglutaminase 2

In the present study the analytical potential of HPLC-MS/MS was utilized for the structural characterization of a post-translational modification of statherin. Human salivary statherin (M(av)5380.0 +/- 0.3 Da) is transformed by the action of transglutaminase 2 into a cyclic derivative with an average molecular mass of 5363.0 +/- 0.3 Da. The intra-molecular bridge is generated by the loss of an ammonia molecule between the unique Ione-pair donating nucleophile Lys-6 and one acceptor among the seven glutamine residues of statherin. Digestion of the cyclic derivative with chymotrypsin, proteinase K, and carboxypeptidase Y, monitored by HPLC-electrospray ionization-ion trap-mass spectrometric analysis, demonstrated that cyclization involved almost specifically Gln-37 (> 95%), with the percentage of Gln-39 implicated in the cross-linkiing being less than 5%. The main derivative was named cyclostatherin Q37. Guineapig transglutaminase 2 showed high affinity for statherin in vitro (Km = 0.65 +/- 0.06 microM). Cyclo-statherin was detected in vivo by HPLC-electrospray ionization ion trap-mass spectrometry analysis of whole human saliva and it accounted for about 1% of total statherin. Detection of cyclo-statherin in whole saliva is suggestive of a putative role of this molecule in the formation of the "oral protein pellicle".

Photooxygenation of chiral 1,3-cyclohexadienes: strong influence of substituents on the stereo- and mode selectivities

Four different chiral 1,3-cyclohexadienes were synthesized and investigated in photooxygenations with singlet oxygen. A strong influence of substituents at the double bond was observed for the mode selectivity of the reactions. Phenyl and alkyl groups afford mixtures of ene and (4 + 2) products, whereas a trimethylsilyl group yields exclusively hydroperoxides, presumably due to a large group effect. Additionally, the same diastereoselectivity for both reaction modes gives evidence for common perepoxide intermediates. Finally, the photooxygenation of a methylensulfonyl substituted 1,3-cyclohexadiene proceeds with very high diastereoselectivity, which can be explained by an intramolecular hydrogen bridge, shielding one face of the compound.

Roles of the Intramolecular Disulfide Bridge in MotX and MotY, the Specific Proteins for Sodium-Driven Motors in Vibrio spp

The proteins PomA, PomB, MotX, and MotY are essential for the motor function of Na+-driven flagella in Vibrio spp. Both MotY and MotX have the two cysteine residues (one of which is in a conserved tetrapeptide [CQLV]) that are inferred to form an intramolecular disulfide bond. The cysteine mutants of MotY prevented the formation of an intramolecular disulfide bond, which is presumably important for protein stability. Disruption of the disulfide bridge in MotX by site-directed mutagenesis resulted in increased instability, which did not, however, affect the motility of the cells. These lines of evidence suggest that the intramolecular disulfide bonds are involved in the stability of both proteins, but only MotY requires the intramolecular bridge for proper function.

Characterization of complexes formed between [Me2Sn(IV)]2+ and carboxymethylcelluloses

Complexes formed between carboxymethylcellulose (CMC) and the [Me2Sn(IV)]2+ cation have been prepared in the solid state and characterized by FTIR and Mössbauer spectroscopy. The complexes contained CMC with varying molar weight and degree of carboxylation, and the complexes were isolated both from acidic and from neutral solutions at varying metal-to-ligand ratios. The characteristic vibration bands of the ligands were identified from their pH-dependent FTIR spectra. In the organotin(IV) complexes obtained at pH∼2, the –COO− moieties were found to be coordinated in a monodentate manner, and the band characteristic of the protonated (unbound) –COO− group(s) was also identified. The broad –OH band can be interpreted as the sum of the contributions of the alcoholic –OH groups of the anhydroglucose units and the mixed organotin aqua complexes. In complexes obtained at pH∼7, the broad –OH band significantly sharpens, which is probably due to the metal-ion induced deprotonation and subsequent coordination of the alcoholic –OH groups. At the same time, –COO− groups are also involved in the coordination of the metal ions, resulting in a complicated network that forms through inter- and intramolecular bridges. Quadrupole splitting (∣Δexp∣) values observed by Mössbauer spectroscopy revealed that the valence state of tin is four in all of the complexes. The ∣Δexp∣ values were compared with the calculated ones, obtained from the pqs theory. From these data, trigonal bipyramidal (Tbp) and octahedral (Oh) geometries have been suggested for the complexes obtained. It has also been concluded that the structure of the complexes prepared depends mainly on the pH of the solution, and it is relatively insensitive to the other parameters, like molar mass or degree of carboxylation of the ligand, or the metal-to-ligand ratio in the reaction mixture.

Brevinin-1BYa: a naturally occurring peptide from frog skin with broad-spectrum antibacterial and antifungal properties

Brevinin-1BYa (FLPILASLAAKFGPKLFCLVTKKC) is a cationic α-helical peptide containing an intramolecular disulphide bridge that is present in skin secretions of the foothill yellow-legged frog Rana boylii. A synthetic replicate of the peptide showed growth inhibitory activity against a range of reference strains of Gram-positive and Gram-negative bacteria, against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) (minimum inhibitory concentration (MIC) = 2.5 μM), and against reference strains and clinical isolates of the opportunistic yeast pathogens Candida albicans, Candida tropicalis, Candida krusei and Candida parapsilosis (MIC ≤ 10 μM). However, the therapeutic potential of the peptide, especially for systemic applications, is restricted by its high haemolytic activity against human erythrocytes (LD 50 = 10 μM). Replacement of the cysteine residues in brevinin-1BYa by serine produced an acyclic analogue with eight-fold reduced haemolytic activity that retained high potency against Gram-positive bacteria, including strains of MRSA (MIC = 5 μM), however activities against Gram-negative bacteria and yeast species were reduced. It is suggested that brevinin-1BYa represents a candidate for drug development, particularly for topical applications against antibiotic-resistant microorganisms.

Design, Syntheses and Extraction Properties of Novel Bis-calix[4]arene and Tris-calix[4]arene Containing Two Kinds of Calix[4]arene Derivative Units

By condensation of calix[4]arene 1,3-bis-hydrazide derivative 5 and calix[4]arene mono-benzaldehyde derivative 2, and then intramolecular bridging with triethylene glycol ditosylates, two novel tris-calix[4]arenes 6 and 7 containing two kinds of calix[4]arene derivative units were prepared in reasonable yield. When reacting compound 5 with calix[4]arene 1,3-bis-benzaldehyde derivative 3, novel bis-calix[4]arene 8 containing two different calix[4]arene derivative units was obtained in high yield. The extraction experiments showed that the new hosts 6, 7 and 8 possessed excellent complexation abilities towards metal cations and α-amino acids. Tris-calix[4]arene 7 showed good complexation selectivity towards Ag+ and basic α-amino acid.

A Blue-Green Absorbing Cross-Linker for Rapid Photoswitching of Peptide Helix Content

Azobenzene derivatives can be used to reversibly photoregulate secondary structure when introduced as intramolecular bridges in peptides and proteins. Here we report the design, synthesis, and characterization of a disubstituted N,N-dialkyl azobenzene derivative that absorbs near 480 nm in aqueous solution and relaxes with a half-life of approximately 50 ms at room temperature. The wavelength of maximum absorbance and the rate of thermal relaxation are solvent-dependent. An increase in the percentage of organic solvent leads, in general, to a blue shift in the absorbance maximum and a slowing of the relaxation rate. In accordance with the design, the thermal relaxation of the azobenzene cross-linker from cis to trans causes an increase in the helix content of one peptide where the linker is attached via cysteine residues spaced at i, i + 11 positions and a decrease in helix content of another peptide with cysteine residues spaced at i, i + 7. This cross-linker design thus expands the possibilities for fast photocontrol of peptide and protein structure.

Monomeric and dimeric GDF-5 show equal type I receptor binding and oligomerization capability and have the same biological activity

Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.

Intermedilysin plays a key role in the invasion of human cells by Streptococcus intermedius

Streptococcus intermedius, which causes endogenous infections leading to abscesses, produces intermedilysin (ILY), a human-specific cytolysin. Because of the significant correlation between higher ILY production levels by S. intermedius and deep-seated abscesses, we constructed an ily knockout mutant and two complementation strains, (i) with the wild-type ily gene and (ii) with an ILY SS-mutant gene (the latter was not cytolytic without reductant due to an intramolecular SS bridge between domains 2 and 3), from ILY high level expressing strain UNS38, in order to investigate the role of ILY in deep-seated infections. Infection with the wild strain and the complementation strain secreting cytolytic ILY reduced the viability of human liver cell line HepG2 at infection but not of rat liver cell line BRL3A, and increased intracellularly concomitantly with increasing HepG2 cell damage. Isogenic ily knockout mutant strain and the complementation strain secreting ILY SS-mutant were not cytotoxic and showed no proliferation in either cell line. Masking of cytotoxic strains with anti-ILY antibody drastically decreased adherence and invasion of HepG2. Moreover, coating ily knockout mutant strain with ILY partially restored adherence to HepG2 but without subsequent bacterial growth. These results indicate that: 1) surface-bound ILY on S. intermedius is an important factor for invasion of human cells by this bacterium; 2) secretion of ILY within host cells is essential for subsequent host cell death; and 3) cell membrane poration by ILY is required in infection steps. These strongly suggest that ILY is an important factor in the pathogenesis of abscesses in vivo by this streptococcus.

The structures of the thrombospondin-1 N-terminal domain and its complex with a synthetic pentameric heparin.

The N-terminal domain of thrombospondin-1 (TSPN-1) mediates the protein's interaction with (1) glycosaminoglycans, calreticulin, and integrins during cellular adhesion, (2) low-density lipoprotein receptor-related protein during uptake and clearance, and (3) fibrinogen during platelet aggregation. The crystal structure of TSPN-1 to 1.8 A resolution is a beta sandwich with 13 antiparallel beta strands and 1 irregular strand-like segment. Unique structural features of the N- and C-terminal regions, and the disulfide bond location, distinguish TSPN-1 from the laminin G domain and other concanavalin A-like lectins/glucanases superfamily members. The crystal structure of the complex of TSPN-1 with heparin indicates that residues R29, R42, and R77 in an extensive positively charged patch at the bottom of the domain specifically associate with the sulfate groups of heparin. The TSPN-1 structure and identified adjacent linker region provide a structural framework for the analysis of the TSPN domain of various molecules, including TSPs, NELLs, many collagens, TSPEAR, and kielin.

Zweifach und dreifach intramolekular verbrückte p‐tert‐Butylcalix[8]aren‐triphosphate – Synthese, Struktur und Stereochemie

AbstractTwo‐ and Threefold Intramolecular Brigdging p‐tert‐Butylcalix[8]arene Triphosphates – Synthesis, Structure and Stereochemistry [1]The phosphorylation of p‐tert‐butylcalix[8]arene (1) with phosphorus pentachloride and hydrolysis gives intramolecular bridging tert‐butylcalix[8]arene triphosphates. The reactivity (esterification, dehydratisation, complexation), the structure (nmr and x‐ray), and the stereochemical behaviour of the phosphates will be discussed.

Alteration of the Factor X Zymogen to Protease Transition Provides Evidence for Allosteric Linkage between the S1 and FVa Binding Sites.

The zymogen to protease transition in the chymotrypsin-like serine protease family follows a well described mechanism in which bond cleavage at a highly conserved site (Arg15-Ile16; chymotrypsin numbering system) results in the unmasking of a new N-terminus that acts as an intramolecular ligand for Asp194. This new salt-bridge drives a conformational change in the so-called “activation domain”, surface loops consisting of the S1 specificity pocket, oxyanion hole, autolysis loop, and sodium biding site. It is well documented in the trypsin system that Ile16-Asp194 internal salt-bridge formation is allosterically linked to the S1 specificity site; that is changes at one site influence the other and vice versa. Blood coagulation factor Xa (FXa) reversibly associates with its cofactor factor Va (FVa) on a membrane surface in the presence of Ca2+ ions with high affinity; an interaction which is not mimicked by the zymogen FX. To determine whether the FX zymogen to protease transition contributes to the expression of a high affinity FVa binding site, we constructed a series of FXa variants which are shifted along this transition pathway. To generate these “zymogen-like” proteins, we made several substitutions at position 16 or 17, with the intent of destabilizing the intramolecular salt bridge to varying degrees. Following a series of preliminary experiments, three mutants were chosen for expression, purification, and activation with RVV-X: I16L, I16G, and V17A. Kinetic studies using peptidyl substrates and active site directed probes revealed that I16L and V17A have an impaired ability to bind these probes (15 to 25-fold increase in the Km or Ki) while the rate of catalysis (kcat) was reduced by 3-fold compared to wild-type FXa (wtFXa; plasma-derived and recombinant). The I16G variant was not inhibited by any of the probes examined and its chromogenic activity was severely impaired (>500 to 1000-fold), precluding calculation of kinetic parameters. These data are consistent with the idea that destabilization of internal salt-bridge formation (Ile16-Asp194) influences binding at the S1 specificity site. In contrast to these results, assembly of I16L and V17A into prothrombinase almost completely restored the Km for peptidyl substrates while the kcat was still 3-fold reduced, indicating that FVa binding can rescue binding at the active site. Surprisingly, even the Km value for I16G was almost completely restored (3-fold increased compared to wtFXa) when assembled in prothrombinase; however a 60-fold reduction in the kcat was found. Consistent with these data, kinetic studies using prothrombin or prethrombin-1 revealed that each of the FXa variants had a normal Km value when assembled in prothrombinase; while the kcat values where reduced to a similar extent as for the chromogenic substrates. Overall our data indicate that direct binding of these FXa variants to FVa rescues binding at S1 site, suggesting allosteric linkage exists between these sites. Thus the FX zymogen to protease transition not only influences the formation of the S1 pocket, but also contributes in a substantial way to the formation of a FVa binding site.