Noncovalent Interactions of the Apple 4 Domain That Mediate Coagulation Factor XI Homodimerization

Abstract

The Apple 4 (A4) domain of human plasma factor XI (FXI) was used to investigate the process of FXI noncovalent dimer formation. Recombinant 6-histidine-tagged A4 domain proteins were prepared utilizing a bacterial expression system. Purification was accomplished under denaturing conditions, followed by a refolding protocol to facilitate correct disulfide bond formation. Analysis of the A4 domain (C321S mutant) by size exclusion chromatography indicated the presence of a slowly equilibrating reversible monomer-dimer equilibrium. The elution profiles reveal highly symmetrical peaks for both dimeric and monomeric species with elution times that were highly reproducible for varying amounts of both the dimeric and monomeric species. The monomer-dimer equilibrium was found to be dependent upon changes in both pH and salt concentration. Under conditions approximating physiologic salt concentration and pH (20 mm HEPES, 100 mm NaCl, and 1 mm EDTA, pH 7.4), it was determined that the monomer-dimer equilibrium was characterized by a dissociation constant (KD) value of 229 ± 26 nm with a calculated ΔG value of 9.1 kcal/mol. This report identifies electrostatic contributions and the presence of a hydrophobic component that mediate interactions at the A4 domain interface. The rate of dissociation for the recombinant A4 domain C321S mutant was examined by monitoring the increase in 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt fluorescence under dissociating conditions, giving a value for a dissociation rate constant (koff) of 4.3 × 10−3s−1. The Apple 4 (A4) domain of human plasma factor XI (FXI) was used to investigate the process of FXI noncovalent dimer formation. Recombinant 6-histidine-tagged A4 domain proteins were prepared utilizing a bacterial expression system. Purification was accomplished under denaturing conditions, followed by a refolding protocol to facilitate correct disulfide bond formation. Analysis of the A4 domain (C321S mutant) by size exclusion chromatography indicated the presence of a slowly equilibrating reversible monomer-dimer equilibrium. The elution profiles reveal highly symmetrical peaks for both dimeric and monomeric species with elution times that were highly reproducible for varying amounts of both the dimeric and monomeric species. The monomer-dimer equilibrium was found to be dependent upon changes in both pH and salt concentration. Under conditions approximating physiologic salt concentration and pH (20 mm HEPES, 100 mm NaCl, and 1 mm EDTA, pH 7.4), it was determined that the monomer-dimer equilibrium was characterized by a dissociation constant (KD) value of 229 ± 26 nm with a calculated ΔG value of 9.1 kcal/mol. This report identifies electrostatic contributions and the presence of a hydrophobic component that mediate interactions at the A4 domain interface. The rate of dissociation for the recombinant A4 domain C321S mutant was examined by monitoring the increase in 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt fluorescence under dissociating conditions, giving a value for a dissociation rate constant (koff) of 4.3 × 10−3s−1. factor XI activated factor XI Apple 4 recombinant Apple 4 polyacrylamide gel electrophoresis isopropyl-β-d-thiogalactopyranoside 3-(cyclohexylamino)propanesulfonic acid 2-(N-morpholino)ethanesulfonic acid nitrilotriacetic acid polymerase chain reaction 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt high performance liquid chromatography fast protein liquid chromatography Human factor XI (FXI)1is a plasma glycoprotein (∼5% carbohydrate) involved in the intrinsic pathway of blood coagulation (1Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1636) Google Scholar). FXI exists as a covalently linked two-chain homodimer, thus making it unique among coagulation factors. Dimeric FXI circulates in plasma as a zymogen in a 1:2 stoichiometric complex with the cofactor high molecular weight kininogen at plasma concentrations in the range of 4–6 μg/ml (∼30 nm) (2Saito H. Goldsmith Jr., G.H. Blood. 1977; 50: 377-385Crossref PubMed Google Scholar, 3Thompson R.E. Mandle Jr., R. Kaplan A.P. J. Clin. Invest. 1977; 60: 1376-1380Crossref PubMed Scopus (200) Google Scholar). Each subunit of the dimer contains 607 amino acids and has a molecular mass of ∼80,000 Da based on reducing SDS gel electrophoresis (4Kurachi K. Davie E.W. Biochemistry. 1977; 16: 5831-5839Crossref PubMed Scopus (119) Google Scholar). The activation of zymogen to be in the presence of a by activated factor and high molecular weight activated FXI (4Kurachi K. Davie E.W. Biochemistry. 1977; 16: 5831-5839Crossref PubMed Scopus (119) Google Scholar, K. Fujikawa K. J. 1991; PubMed Google Scholar). dependent upon of the bond and K. Davie E.W. Biochemistry. PubMed Scopus Google Scholar). Each subunit of of a and chain disulfide (4Kurachi K. Davie E.W. Biochemistry. 1977; 16: 5831-5839Crossref PubMed Scopus (119) Google Scholar). The chain as a involved in the activation of coagulation K. H. Davie E.W. Biochemistry. PubMed Scopus Google Scholar). The chain of contains Apple of amino acids R. Davie E.W. Biochemistry. PubMed Scopus Google Scholar). Each of the Apple contains disulfide and the and Apple Fujikawa K. Davie E.W. Biochemistry. 1991; 30: PubMed Scopus Google Scholar). in the A4 domain has in the of a FXI dimer Fujikawa K. Davie E.W. Biochemistry. 1991; 30: PubMed Scopus Google Scholar). The Apple domain contains for cofactor high molecular weight for and for the domain of J. PubMed Google Scholar, J. PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google the contains a J. 1991; PubMed Google the has in interactions with with and with J. PubMed Scopus Google Scholar, K. J. PubMed Scopus Google Scholar, Biochemistry. Scopus Google Scholar, J. PubMed Scopus Google and the (A4) contains activated factor with to mediate dimer Fujikawa K. Davie E.W. Biochemistry. 1991; 30: PubMed Scopus Google Scholar, J. PubMed Google Scholar, Davie E.W. Biochemistry. PubMed Scopus Google Scholar). of the FXI and that to physiologic in blood FXI activated on the and the K. H. Davie E.W. Biochemistry. PubMed Scopus Google Scholar). The recombinant domain of FXI to the activated with the as that found for dimeric that for the with the Biochemistry. Scopus Google Scholar). it to J. PubMed Scopus Google J. 1991; PubMed Google domain of FXI to both and to the it has that the domain subunit of the dimer the the domain of the subunit with Blood. PubMed Google that FXI in the A4 domain of FXI was with that of in with dimeric were in a utilizing as a with with the the monomeric to the domain for with and be in to FXI be for to at of the activation of by that activated a reaction of a Biochemistry. PubMed Scopus Google Scholar). the that the dimer a the for of the that it be for FXI to as a dimeric the of FXI to the and involved in Davie E.W. Biochemistry. PubMed Scopus Google that at of the molecular for dimer the to the A4 domain of with the A4 domain of FXI for the and factor as as by gel Davie E.W. Biochemistry. PubMed Scopus Google Scholar). the as as the FXI the of with in that as as determined by gel Davie E.W. Biochemistry. PubMed Scopus Google Scholar). that the A4 mediate noncovalent interactions in dimer by a in at Fujikawa K. Davie E.W. Biochemistry. 1991; 30: PubMed Scopus Google Scholar, Davie E.W. Biochemistry. PubMed Scopus Google Scholar). prepared the domain of FXI as as a C321S mutant to dimer and to of under conditions of varying pH and a gel to that by K. PubMed Scopus Google for the of and recombinant dissociation and and and were used for were and were and protein molecular mass were and the were was HEPES, and the gel molecular mass were the acid the gel and the polymerase chain reaction were The was was The and were × and × were The the and the were The protein and the were and were by was The for of was The 4,4′-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt was The domain contains that for The was by utilizing and FXI as a the FXI a W. and W. of of to a that to that for the A4 domain of that of the A4 domain were prepared by The contains with a was used to The contains with a to The of the as The of the as The was as of FXI as a 1 of both the and of of reaction and at mm to a of 100 The reaction was with 100 of to The reaction were in a The were to for followed by at for 1 for 1 and for The reaction was by at for and a at 4 The was with the was in the of the C321S mutant The protocol was PubMed Scopus Google Scholar). was a human FXI A4 domain a expression The reaction a 1 of a a and a The as The in the was in the to thus to be by in the mutant amino acid The of nm) of the and of of reaction and at mm to a of of were for for 1 for 1 and for The reaction was by at for with a at 4 of of the to the and of of the The protocol as in 1 used in and The was with the by The was to the and were the was by the of expression were in in the presence of 100 μg/ml and μg/ml at The were The were of protein expression the of mm followed by and at for were by × for and as at The concentration of protein was determined by at nm of was used to and to noncovalent of at were in and pH at of of and to at for 1 The was the at × for at 4 The was with a and The was to at for 1 The was a of of and pH a with and pH the at nm was and of and pH The FXI domain was with of and pH protein were by The protein was a protocol that of a in as the molecular mass reducing Biochemistry. PubMed Scopus Google Scholar). concentration was in the range of with a was The of mm 100 and mm at pH of changes was used to the and concentrations and 1 mm and mm and and 100 and and of the FXI domain was The protein was to a of and the protein concentration to was under reducing conditions was used to and under conditions to FXI The was by with proteins at the of the to that at the of the FXI of mm and 100 mm NaCl, pH of to the FXI domain was The reaction was at for of the of was determined by the of the FXI domain the FXI domain was the The was with by of a The reaction was to the followed by a (20 mm and 100 mm at pH The FXI domain with was with mm in the system. The in the of gel chromatography to the FXI domain and to protein that gel was in mm and 100 mm NaCl, pH The proteins were at a rate of 1 and protein elution was by at that at a of the dimeric FXI domain were and with a the of the for a were the gel molecular mass were for the presence of the The protein was examined by The domain was examined by and mass by the at the of The the presence of a species of protein with a molecular mass of with the calculated molecular mass of the was used to R. R. indicated of of fast protein liquid chromatography was used with high × to for the FXI domain C321S was in and of dimer formation. the of the for a were at varying pH and salt concentrations the gel molecular mass proteins were with molecular as and The of varying protein and salt concentration were used to pH mm 100 mm NaCl, and 1 mm for the pH were as pH pH and HEPES, pH and and pH used to salt mm HEPES, the concentration and 1 mm EDTA, pH the proteins were the for in The protein was of by and protein concentration was determined by at nm with the were prepared by of and for at to that equilibrium was of 100 at protein concentrations were the of protein species was in the as the at a rate of 1 The elution was followed by at The were The times were determined by of elution peaks at and The calculated under the elution peaks was used to the and of dimeric and monomeric species in the of the dimeric as a of the protein concentration was used to dimer determined were by a factor of ± The factor was of the protein as the protein the The for be found under and in K. PubMed Scopus Google and J. PubMed Google The factor the at by the K. PubMed Scopus Google Scholar, J. PubMed Google Scholar). The of dissociation was calculated the ΔG the constant × × the at were and the dissociation constant the concentration at amounts of dimer and in were on The of were and the concentration was determined by at nm of Biochemistry. PubMed Scopus Google Scholar). amounts of the were used to the domain monitoring concentrations were constant at The were at and was at The and were to 4 and were with to of and protein The of dissociation was followed in a by monitoring the increase in fluorescence as dissociation the protein was to a concentration of in the presence of The were the and a with a rate The rate constant for the of to dimer was calculated the the dissociation constant determined by size exclusion chromatography and the dissociation rate constant determined by fluorescence the FXI domain as and facilitate the of the 6-histidine-tagged protein in to the A4 domain of FXI was and in the expression The contains a expression to be by the of the expression of the domain with and with and in the presence of the and the as a molecular to facilitate of the recombinant protein by a high for the at pH with in pH for the of proteins The protein of was at pH a the of the and the with the The was in and by both at nm and The FXI domain in the of This to be to the protein the in with was by the by and with The the chromatography a was of The protein was a protocol that of a in as the molecular mass reducing Biochemistry. PubMed Scopus Google Scholar). of the the protein slowly the correct to in of the presence of for a disulfide of disulfide for of the disulfide the protein concentration the was a of protein was at to formation. of the protein was making it the in the dependent upon disulfide bond highly to in and protein concentration R. R. Scholar, W. H. for the and the of protein at The protein that was the by and of was examined by under reducing and conditions and gel the of protein at protein that was of as by by and peaks on gel dimer at that protein was a range of species to as and upon as as elution peaks upon gel chromatography R. R. Scholar, W. H. Scholar). of protein based on the chromatography were in the of The of the was accomplished by with of the of the the amino acids the The molecular mass of the protein was by Da to a calculated molecular mass of The reaction was by the FXI domain the the FXI the was to a with The the A4 and the A4 (20 mm and 100 mm NaCl, pH species on the was and the A4 domain of both and the A4 domain of to a at pH that proteins at This for a with the to the of the A4 domain was accomplished by of mm to the were and by at nm and and the A4 domain in a in the the protein was on a gel The gel accomplished of and of the A4 domain protein that of that at to the dimer of the FXI domain were were with a of molecular mass elution by chromatography of the gel molecular mass The were with the to of the A4 was determined by was the gel that the A4 domain was to 1 on the protein the chromatography a of of FXI domain of expression The for both and noncovalent dimer were molecular mass of both and were by to molecular of and size of The domain as a dimer molecular mass of and size of The mutant as a to that of the The that the domain exists as a dimer and that dimer by a disulfide as be The mutant dimer a at was of under conditions, as the gel Under conditions, SDS was of the C321S dimer that the mutant dimer noncovalent amino acid high × was used to a to monomer-dimer for mutant of by both K. PubMed Scopus Google and and R.E. Biochemistry. PubMed Scopus Google to dissociation of and recombinant and the monomer-dimer equilibrium of the of the for a were under varying conditions the gel molecular mass determined by was used to the dimeric and monomeric species for of dimeric as a of the concentration to to the a dissociation for the C321S mutant with elution profiles in The elution of the peaks dimer and in to molecular of and to the calculated and dimer of the Under conditions approximating physiologic salt concentration and pH (20 mm HEPES, 100 mm NaCl, and 1 mm EDTA, pH 7.4), it was determined that the monomer-dimer equilibrium was characterized by of ± This value of for a factor the gel K. PubMed Scopus Google Scholar, J. PubMed Google to a value of 229 The for in the under and in K. PubMed Scopus Google and J. PubMed Google The that the monomer-dimer equilibrium of as by a in the of dimer to as a of protein concentration. The of the equilibrium was by the dimer to a of chromatography and the presence of peaks to dimer and The equilibrium be to dimeric monomeric that the protein in the reaction and in the equilibrium The elution times were highly reproducible for both varying amounts of the dimeric and monomeric as by at elution profiles as highly of a of monomeric and dimeric in equilibrium species and Scholar). The of interactions with the that the A4 domain by to and that the disulfide bond at C321S for dimer formation. with the of Davie E.W. Biochemistry. PubMed Scopus Google for the mutant of as a dimeric upon gel the that the C321S mutant exists in a reversible monomer-dimer equilibrium that be and used to for under varying for dissociation the size exclusion chromatography was examined of the domain was to and in mm HEPES, 100 mm NaCl, and 1 mm EDTA, pH were on a at times The were to that the equilibrium was the that equilibrium to The concentration for and equilibrium was and was with determined of of at concentrations of and be and This This to that the protein in the equilibrium of to the dissociation of the dimer the dimer size exclusion chromatography at concentrations of was used to a of pH in a range and of the dissociation in The in The 229 nm) for the dimer was in the pH range pH ± ΔG to pH 229 ± 26 ΔG 9.1 was for the a was pH with pH 229 ± 26 nm and ΔG 9.1 ± nm and ΔG increase in was followed by a increase in at pH ± ΔG increase in pH a of the dimer in increase in pH to The as a of pH for a at the dimer that in dimer formation. in pH and the of amino acid with a of of pH on dimer ± of ± ± ± ± ± the of pH on dimer dissociation in and The were determined of dimer utilizing and were for of on the as under The of dissociation was calculated the ΔG the constant × × the and the dimer dissociation constant The ± of in a the of pH on dimer dissociation in and The were determined of dimer utilizing and were for of on the as under The of dissociation was calculated the ΔG the constant × × the and the dimer dissociation constant was for pH and that the range in The of and in elution it was to the that the A4 domain be in pH conditions and changes in the protein changes in the rate of dissociation with the of salt on dimer of ± ± of ± ± 26 ± of ± ± of ± ± of ± ± ± of ± the of salt on dimer dissociation in of and The were determined of dimer utilizing and were for of on the as under The of dissociation was calculated the ΔG the constant × × the and the dimer dissociation constant The ± of The in a the of salt on dimer dissociation in of and The were determined of dimer utilizing and were for of on the as under The of dissociation was calculated the ΔG the constant × × the and the dimer dissociation constant the dimer size exclusion chromatography at concentrations of was used to dissociation as a of salt concentration and of the dissociation in The in the concentrations of and the to 229 ± 26 nm and ΔG 9.1 were increase in salt concentration in in ± ΔG salt concentration of ± ΔG the and be of increase in salt concentration a of the dimer as by the in as the concentration was to The the of salt on the dimer with the that electrostatic interactions to FXI domain concentrations increase in times for both the dimeric and monomeric species. that protein by the as a of interactions with the changes in to the of hydrophobic interactions in A4 dimer formation. The was used to the A4 domain monitoring to a hydrophobic hydrophobic on the of a a increase in fluorescence PubMed Scopus Google Scholar). with nm) the and in a upon of the to a hydrophobic Biochemistry. PubMed Scopus Google Scholar). The of both the fluorescence increase and the in dependent upon the of the J. PubMed Scopus Google Scholar). increase in the fluorescence and a in the to nm were upon of of to concentrations of were in the of a K. J. PubMed Scopus Google Scholar). a the fluorescence of in the of was with the fluorescence of in the presence of protein the domain and the domain in the presence of amounts of and the in fluorescence of the to the mutant domain with a increase in as by a that was in The of the of a with a to the covalently dimeric domain to the as by the of a increase in the of hydrophobic on the of the covalently linked the of the on the a domain in was with The a of of the as by the in The on the of the domain be by of the protein to that the hydrophobic in the presence of the dimer that a hydrophobic upon dimer dissociation and that be by of the hydrophobic on the monomeric subunit to the that a hydrophobic component exists at the be in for A4 dimer formation. The in the fluorescence of upon to the domain was for monitoring the of dissociation of both the and were to a concentration of in the presence of and for in increase in fluorescence for a reaction with a dissociation rate constant (koff) of 4.3 × for the dissociation of dimer to the of 229 ± 26 nm exclusion were to rate constant for the of to dimer of × The of the with the domain monomer-dimer equilibrium a at the the dimer of the domain as as the noncovalent of FXI as a of to that based on the that to the at a rate with the rate of dissociation of the the rate of of a with a for a reaction a rate constant in the range of and in to and W. H. Scholar). that the rate of for the were by for changes in protein upon of the with the the rate of of the at to the protein be with rate for dimer dissociation of 4.3 × in the dissociation of the dimer and the of to the the covalently linked protein the the of the for the dimer interface. in with the in increase in fluorescence in the presence of the The of both the fluorescence of and the gel on the determined rate of dissociation 4.3 × calculated a the dissociation reaction of the of the gel in the of the were at of the equilibrium to This as to in in concentration and a for of to for the of protein the gel The in the of the noncovalent dimer of FXI by of a A4 report nm) and the of dissociation for the noncovalent mutant under physiologic conditions, as determined by size exclusion report of 4.3 × and a calculated of × fluorescence The FXI domain and the mutant and to both and noncovalent of the recombinant mass of the protein and the of disulfide bond that in a protein the domain with a of R. R. Scholar, W. H. in the The of that proteins the that to the domain exists as a species upon mass and gel the that the protein and J. PubMed Scopus Google Scholar). proteins for to and were to the as by the disulfide bond in Fujikawa K. Davie E.W. Biochemistry. 1991; 30: PubMed Scopus Google to for the A4 domain the a of proteins and in that Scholar). that the domain were by the that of the domain in chromatography size exclusion chromatography has in the and has a for equilibrium for and Scholar, J. PubMed Google Scholar, Biochemistry. PubMed Scopus Google Scholar). by to be to equilibrium and fluorescence among to K. PubMed Scopus Google Scholar, R.E. Biochemistry. PubMed Scopus Google Scholar). The in for a process in a of both dimeric and monomeric by the chromatography process and exists in a slowly equilibrium and Scholar). a slowly the process the reversible of dimer and This highly symmetrical peaks in and Scholar, Biochemistry. PubMed Scopus Google Scholar). examined the of pH on the dissociation equilibrium the mutant and increase at the pH the pH be to of on the protein in a of the a high of and of that the proteins both in to and of with the and Scholar). that the pH to a of a amino acid chain involved in the dimer The changes in in the pH range to increase in of a chain with a value of and the amino Biochemistry. W. H. Scholar, and W. H. Scholar). the A4 domain in both the FXI Fujikawa K. Davie E.W. Biochemistry. 1991; 30: PubMed Scopus Google and protein in disulfide to mediate dimer formation. The amino of the A4 domain FXI in a bond with both the FXI and of with and the the were at the dimer to the and a for of the it highly that the of the subunit a in the dimer to the of that increase in pH the of the A4 domain to the that of amino acid chain with a to the The that electrostatic component dimer a chain to be it of that the the of proteins a and the for to the of the FXI dimer interface. has to a in the of a of protein J. PubMed Scopus Google Scholar). of by in electrostatic to subunit interactions by Biochemistry. PubMed Scopus Google H. PubMed Scopus Google Scholar). The of and has that by R. R. K. Google Scholar, PubMed Scopus Google Scholar, J. 1991; PubMed Google Scholar). interactions in the of FXI by the A4 The in equilibrium at the high concentrations of salt to be a of by the of and a in the interactions at the Biochemistry. PubMed Scopus Google Scholar, J. PubMed Google Scholar). as in the to in the of that in PubMed Scopus Google Scholar). the increase in salt concentration the interactions by the at the A4 dimer interface. Under conditions of high to be at protein PubMed Scopus Google Scholar, and Google hydrophobic in a in ΔG to salt that the increase in salt hydrophobic at the dimer for the to of at the interface. The of the for hydrophobic on the of proteins 1991; PubMed Scopus Google has to the of protein found in coagulation cofactor K. J. PubMed Scopus Google Scholar). The the of a hydrophobic on as as the of hydrophobic on the covalently linked The presence of a on the of in formation. of a hydrophobic component at the A4 dimer and that the to a in with the changes in that the hydrophobic component for the by salt has for the of FXI as as for the of proteins in with of that that the of interactions and and interactions to to J. J. PubMed Scopus Google Scholar). The of the a of hydrophobic and the PubMed Scopus Google Scholar). This of the the monomeric of FXI and that of coagulation and of FXI Google Scholar). the dimeric of FXI be for to on the Biochemistry. Scopus Google Scholar, Blood. PubMed Google Scholar, Biochemistry. PubMed Scopus Google it to the of FXI to FXI with the proteins to of the the A4 be to the domain with the FXI C321S mutant to of the FXI be by the of as fluorescence Biochemistry. PubMed Scopus Google Scholar, PubMed Scopus Google and K. H. K. K. J. PubMed Scopus Google that a equilibrium that of and species. with and with to amino acid contributions at the dimer interface. to and for and and to for in