Recombinant PAS-heme domains of oxygen sensing proteins: high level production and physical characterization

Details of a high-level recombinant production method for the heme-PAS domains of heme oxygen sensing proteins from Sinorhizobium meliloti (Sm) (formerly Rhizobium meliloti, Rm), Bradyrhizobium japonicum (Bj), and Escherichia coli (Ec) are described. Using a newly proposed, concise, and unambiguous naming system (also described here) these proteins are: SmFixLH(128-264), BjFixLH(140-270), and EcDosH(1-147). In addition, high-level production of BjFixL(140-505), the soluble full-length protein containing both heme (oxygen sensing) and kinase (catalytic) domains is described. Using an IPTG-inducible pET/BL21 expression system and a rapid, two-column purification has resulted in increased yields of 3- to 17-fold over literature values. The recombinant proteins are highly pure as judged by SDS-PAGE, MALDI-TOF mass spectrometry, and a UV-visible purity index. To our knowledge, this work includes the first mass spectrometry analysis of any PAS-heme protein and provides high-resolution confirmation of each protein's identity. These production and characterization improvements make possible future spectroscopic and dynamics studies designed to elucidate the intramolecular/interdomain signal that follows heme-domain oxygen dissociation.     

Origins of aging mass loss in recombinant N-terminus and C-terminus deletion mutants of the heme-PAS biosensor domain BjFixLH(140-270)

Nine recombinant FixL heme domains from Bradyrhizobium japonicum previously were shown to exhibit mass instability independent of many environmental factors (J.D. Satterlee, C. Suquet, A. Bidwai, J. Erman, L. Schwall, R. Jimenez, Biochemistry 47 (2008) 1540-1553). Two of those recombinant proteins were produced in remote laboratories. Mass losses begin appearing at completion of isolation and comprise a substantial proportion of samples within 1-3 days of storage and handling. Thus, degradation occurs during the time frame of experiments and crystallization. Detailed understanding of this instability is desired in order to formulate stable heme-PAS sensor domains for experimentation and for a mechanistic interpretation. However, mass spectra of the full length heme-PAS domain, BjFixLH_140-270, are complex by 1-3 days following isolation due to broad features and a high density of overlapping peaks, so that individual peak assignments are at present ambiguous. This stymies direct, quantitative interpretation of the source of the observed mass losses. To solve this dilemma amino-terminal primary sequencing and MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight) mass spectrometry monitoring of three terminal variants of BjFixLH_140-270 have been achieved. The working hypothesis, that the experimentally observed mass losses originate in the PAS protein sequence termini, has been substantiated. This establishes a basis for interpreting the more complex results from aging full length BjFixLH_140-270.     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.     

Heterologous expression of an endogenous rat cytochrome b(5)/cytochrome b(5) reductase fusion protein: identification of histidines 62 and 85 as the heme axial ligands

The gene coding for expression of an endogenous soluble fusion protein comprising a b-type cytochrome-containing domain and a FAD-containing domain has been cloned from rat liver mRNA. The 1461-bp hemoflavoprotein gene corresponded to a protein of 493 residues with the heme- and FAD-containing domains comprising the amino and carboxy termini of the protein, respectively. Sequence analysis indicated the heme and flavin domains were directly analogous to the corresponding domains in microsomal cytochrome b(5) (cb5) and cytochrome b(5) reductase (cb5r), respectively. The full-length fusion protein was purified to homogeneity and demonstrated to contain both heme and FAD prosthetic groups by spectroscopic analyses and MALDI-TOF mass spectrometry. The cb5/cb5r fusion protein was able to utilize both NADPH and NADH as reductants and exhibited both NADPH:ferricyanide (k(cat) = 21.7 s(-1), K(NADPH)(m) = 1 microM. K(FeCN6)(m) = 8 microM) and NADPH:cytochrome c (k(cat) = 8.3 s(-1), K(NADPH)(m) = 1 microM. K(cyt c)(m) = 7 microM) reductase activities with a preference for NADPH as the reduced pyridine nucleotide substrate. NADPH-reduction was stereospecific for transfer of the 4R-proton and involved a hydride transfer mechanism with a kinetic isotope effect of 3.1 for NADPH/NADPD. Site-directed mutagenesis was used to examine the role of two conserved histidine residues, H62 and H85, in the heme domain segment. Substitution of either residue by alanine or methionine resulted in the production of simple flavoproteins that were effectively devoid of both heme and NAD(P)H:cytochrome c reductase activity while retaining NAD(P)H:ferricyanide activity, confirming that the former activity required a functional heme domain. These results have demonstrated that the rat cb5/cb5r fusion protein is homologous to the human variant and has identified the heme and FAD as the sites of interaction with cytochrome c and ferricyanide, respectively. Mutagenesis has confirmed the identity of both axial heme ligands which are equivalent to the corresponding residues in microsomal cytochrome b(5).     

New mechanistic insights from structural studies of the oxygen-sensing domain of Bradyrhizobium japonicum FixL

The FixL heme domain serves as the dioxygen switch in the FixL/FixJ two-component system of Rhizobia. Recent structural studies of the Bradyrhizobium japonicum FixL heme domain (BjFixLH) have suggested an allosteric mechanism that is distinct from the classical hemoglobin model. To gain further insight into the FixL sensing mechanism, structures of BjFixLH bound to dioxygen, imidazole, and nitric oxide have been determined. These structures, particularly the structure of BjFixLH bound to its physiological ligand, dioxygen, have helped to address a number of important issues relevant to the BjFixLH sensing mechanism. On the basis of the oxy-BjFixLH structure, a conserved arginine is found to stabilize the dioxygen ligand in a mode reminiscent of the distal histidine in classical myoglobins and hemoglobins. The structure of BjFixLH bound to imidazole elucidates the structural requirements for accommodating sterically bulky ligands. Finally, the structure of BjFixLH bound to nitric oxide provides evidence for a structural intermediate in the heme-driven conformational change.     

Characterization of conformational changes coupled to ligand photodissociation from the heme binding domain of FixL

Using transient absorption spectroscopy and photoacoustic calorimetry (PAC), we have characterized carbon monoxide photodissociation and rebinding to two forms of the heme domain of Bradyrhizobium japonicum FixL. Transient absorption results for the complete heme domain (FixL residues 140-270) and a truncated heme domain (missing 11 residues on the N-teminal end and 14 amino acid residues on the C-terminal end of the full length heme domain) show similar rates for ligand rebinding to the five-coordinate heme domain and the absence of any transient intermediate on a microsecond time scale. Results from PAC studies show that both the truncated and complete heme domains undergo a contraction upon ligand photolysis. In addition, CO photolysis from the complete heme domain gives rise to an intermediate with a lifetime of approximately 150 ns which is absent in the truncated heme domain. We attribute the 150 ns phase to ligand release to the solvent which may be accelerated in the case of the truncated domain. The initial contraction is attributed to changes in the charge distribution due to reorganization of the surface salt bridge formed between Glu182 and Arg227 or possibly to reorientation of Arg206. Changes in the charge distribution may play an important role in communication between the sensor domain and the regulatory domain and thus may be part of the signal transduction pathway.     

Recombinant human erythropoietin with additional processable protein domains: Purification of protein synthesized in <Emphasis Type="Italic">Escherichia coli</Emphasis> heterologous expression system

Three variants of human recombinant erythropoietin (rhEPO) with additional N-terminal protein domains were obtained by synthesis in an Escherichia coli heterologous expression system. These domains included (i) maltose-binding protein (MBP), (ii) MBP with six histidine residues (6His) in N-terminal position, (iii) s-tag (15-a.a. oligopeptide derived from bovine pancreatic ribonuclease A) with N-terminal 6His. Both variants of the chimeric protein containing MBP domain were prone to aggregation under nondenaturing conditions, and further purification of EPO after the domain cleavage by enterokinase proved to be impossible. In the case of 6His-s-tag-EPO chimeric protein, the products obtained after cleavage with enterokinase were successfully separated by column chromatography, and rhEPO without additional domains was obtained. Results of MALDI-TOF mass spectrometry showed that after refolding 6His-s-tag-EPO formed a structure similar to that of one of native EPO with two disulfide bonds. Both 6His-s-tag-EPO and rhEPO without additional protein domains purified after proteolysis possessed the same biological activity in vitro in the cell culture.     

Characterization of Drosophila nitric oxide synthase: a biochemical study

The heme and flavin-binding domains of Drosophila nitric oxide synthase (DNOS) were expressed in Escherichia coli using the expression vector pCW. The denatured molecular mass of the expressed protein was 152kDa along with a proteolytically cleaved product of 121kDa. The DNOS heme protein exhibited very low Ca(2+)/calmodulin-dependent NO synthase activity. The trypsin digestion patterns were different from nNOS. The full-length DNOS protein had high degree of stability against trypsin. The activity assay of trypsin-digested protein confirmed the same result. Urea dissociation profile of DNOS full-length protein showed that the reductase domain activity was much more susceptible towards urea than the oxygenase domain activity. Urea gradient gel of DNOS full-length protein established distinct transition of dissociation and unfolding in the range 3-4M urea. Reductase domain activity of full-length DNOS protein against external electron acceptors like cytochrome c indicated slow electron transfer from FMN. The bacterial expression of DNOS full-length protein represents an important development in structure-function studies of this enzyme and comparison with other mammalian NOS enzymes which is evolutionary significant.     

Heme binding in the NEAT domains of IsdA and IsdC of Staphylococcus aureus

Absorption, magnetic circular dichroism (MCD), and electrospray mass spectral (ESI-MS) data are reported for the heme binding NEAr iron Transporter (NEAT) domains of IsdA and IsdC, two proteins involved in heme scavenging by Staphylococcus aureus. The mass spectrometry data show that the NEAT domains are globular in structure and efficiently bind a single heme molecule. In this work, the IsdA NEAT domain is referred to as NEAT-A, the IsdC NEAT domain is referred to as NEAT-C, heme-free NEAT-C is NEAT-A and NEAT-C are inaccessible to small anionic ligands. Reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-A results in coordination by histidine and opens access, allowing for CO axial ligation, yielding 6-coordinate low-spin Fe(II) heme. In contrast, reduction of the high-spin Fe(III) heme iron to 5-coordinate high-spin Fe(II) in NEAT-C results in loss of the heme from the binding site of the protein due to the absence of a proximal histidine. The absorption and MCD data for NEAT-A closely match those previously reported for the whole IsdA protein, providing evidence that heme binding is primarily a property of the NEAT domain.     

Molecular structure and properties of lectin from tomato fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro(4) motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

基质辅助激光解吸电离飞行时间质谱技术作为常见益生菌菌种鉴定辅助工具的研究

目的评价基质辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)技术用于常见益生菌菌株鉴定及潜在益生菌菌株筛选的可行性。方法利用16S rDNA序列分析在方法学上对MALDI-TOF MS技术的鉴定能力进行研究;通过MALDI-TOF MS技术对现有保藏菌株的鉴定结果研究MALDI-TOF MS技术的鉴定准确性及优越性。结果 MALDI-TOF MS技术具备较16S rDNA序列分析更高的菌株鉴定能力;MALDI-TOF MS技术的鉴定结果准确、稳定。结论 MALDI-TOF MS技术可以作为准确、快速、廉价及可高通量操作的菌株鉴定方法应用于常见益生菌菌株的鉴定及潜在益生菌菌株的筛选。     

Relationships between heme incorporation, tetramer formation, and catalysis of a heme-regulated phosphodiesterase from Escherichia coli: a study of deletion and site-directed mutants

The heme-regulated phosphodiesterase (PDE) from Escherichia coli (Ec DOS) is a tetrameric protein composed of an N-terminal sensor domain (amino acids 1-201) containing two PAS domains (PAS-A, amino acids 21-84, and PAS-B, amino acids 144-201) and a C-terminal catalytic domain (amino acids 336-799). Heme is bound to the PAS-A domain, and the redox state of the heme iron regulates PDE activity. In our experiments, a H77A mutation and deletion of the PAS-B domain resulted in the loss of heme binding affinity to PAS-A. However, both mutant proteins were still tetrameric and more active than the full-length wild-type enzyme (140% activity compared with full-length wild type), suggesting that heme binding is not essential for catalysis. An N-terminal truncated mutant (DeltaN147, amino acids 148-807) containing no PAS-A domain or heme displayed 160% activity compared with full-length wild-type protein, confirming that the heme-bound PAS-A domain is not required for catalytic activity. An analysis of C-terminal truncated mutants led to mapping of the regions responsible for tetramer formation and revealed PDE activity in tetrameric proteins only. Mutations at a putative metal-ion binding site (His-590, His-594) totally abolished PDE activity, suggesting that binding of Mg2+ to the site is essential for catalysis. Interestingly, the addition of the isolated PAS-A domain in the Fe2+ form to the full-length wild-type protein markedly enhanced PDE activity (>5-fold). This activation is probably because of structural changes in the catalytic site as a result of interactions between the isolated PAS-A domain and that of the holoenzyme.     

Molecular Structure and Properties of Lectin from Tomato Fruit

A cDNA encoding tomato fruit lectin was cloned from an unripe cherry-tomato fruit cDNA library. The isolated lectin cDNA contained an open reading frame encoding 365 amino acids, including peptides that were sequenced. The deduced sequence consisted of three distinct domains: (i) an N-terminal short extensin-like domain; (ii) a Cys-rich carbohydrate binding domain composed of four almost identical chitin-binding domains; (iii) an internal extensin-like domain of 101 residues containing 15 SerPro₄ motifs inserted between the first and second chitin-binding domains. The molecular weight of the lectin was 65,633 and that of the deglycosylated lectin was 32,948, as determined by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This correlated with the estimated molecular weight of the deduced sequence. Recombinant tomato lectin expressed in Pichia pastoris possessed chitin-binding but not hemagglutinating activity. These findings confirmed that the cDNA encoded tomato lectin.     

Human von Willebrand factor: a multivalent protein composed of identical subunits

A large-scale method for the isolation of von Willebrand factor (vWF) from human factor VIII concentrates was developed in order to study the structure of this protein and its platelet binding activity. vWF is composed of a number of glycoprotein subunits that are linked together by disulfide bonds to form a series of multimers. These multimers appear to contain an even number of subunits of 270K. Two minor components of Mr 140K and 120K were also identified, but these chains appear to result from minor proteolysis. The smallest multimer of vWF contained nearly equimolar amounts of the 270K, 140K, and 120K subunits, while the largest multimers contained less than 20% of the two minor components. Amino acid sequence analysis, amino acid composition, and cleavage by cyanogen bromide indicate that the 270K subunits are identical and each is a single polypeptide chain with an amino-terminal sequence of Ser-Leu-Ser-Cys-Arg-Pro-Pro-Met-Val-Lys and a carboxyl-terminal sequence of Glu-Cys-Lys-Cys-Ser-Pro-Arg-Lys-Cys-Ser-Lys. Platelet binding in the presence of ristocetin was 8-fold greater with multimers larger than five (i.e., containing more than 10 subunits of 270K) as compared to multimers less than three (containing less than six subunits of 270K). However, partially reduced vWF (Mr 500K), regardless of whether it was prepared from large or small molecular weight multimers, gave platelet binding similar to that of the smallest multimers. Likewise, partial proteolysis by elastase, thermolysin, trypsin, or chymotrypsin produced small "multimer-like" proteins with platelet binding properties similar to either partially reduced vWF or to the smallest multimers. We conclude that human vWF contains identical 270K subunits assembled into a multivalent structure. Disassembly by either partial reduction or partial proteolysis produces essentially monovalent protein with platelet binding properties similar to that of the smallest multimers. Multivalency is likely the primary factor responsible for the increase in biological activity with multimer size.     

Heme Ligand Binding Properties and Intradimer Interactions in the Full-length Sensor Protein Dos from Escherichia coli and Its Isolated Heme Domain

Dos from Escherichia coli is a bacterial gas sensor protein comprising a heme-containing gas sensor domain and a phosphodiesterase catalytic domain. Using a combination of static light scattering and gel filtration experiments, we established that, as are many other sensor proteins, the full-length protein is dimeric. The full-length dimer (association constant <10 nm) is more stable than the dimeric heme domain (association constant ∼1 μm), and the dimer interface presumably includes both sensor and catalytic domains. Ultrafast spectroscopic studies showed little influence of the catalytic domain on kinetic processes in the direct vicinity of the heme. By contrast, the properties of ligand (CO and O₂) binding to the heme in the sensor domain, occurring on a microsecond to second time scale, were found to be influenced by (i) the presence of the catalytic domain, (ii) the dimerization state, and in dimers, (iii) the ligation state of the other subunit. These results imply allosteric interactions within dimers. Steady-state titrations demonstrated marked cooperativity in oxygen binding to both the full-length protein and the isolated heme domain, a feature not reported to date for any dimeric sensor protein. Analysis of a variety of time-resolved experiments showed that Met-95 plays a major role in the intradimer interactions. The intrinsic binding and dissociation rates of Met-95 to the heme were modulated ∼10-fold by intradimer and sensor-catalytic domain interactions. Dimerization effects were also observed for cyanide binding to the ferric heme domains, suggesting a similar role for Met-95 in ferric proteins.     

The N-terminus of apolipoprotein A-V adopts a helix bundle molecular architecture

Previous studies of recombinant full-length human apolipoprotein A-V (apoA-V) provided evidence of the presence of two independently folded structural domains. Computer-assisted sequence analysis and limited proteolysis studies identified an N-terminal fragment as a candidate for one of the domains. C-Terminal truncation variants in this size range, apoA-V(1-146) and apoA-V(1-169), were expressed in Escherichia coli and isolated. Unlike full-length apoA-V or apoA-V(1-169), apoA-V(1-146) was soluble in neutral-pH buffer in the absence of lipid. Sedimentation equilibrium analysis yielded a weight-average molecular weight of 18811, indicating apoA-V(1-146) exists as a monomer in solution. Guanidine HCl denaturation experiments at pH 3.0 yielded a one-step native to unfolded transition that corresponds directly with the more stable component of the two-stage denaturation profile exhibited by full-length apoA-V. On the other hand, denaturation experiments conducted at pH 7.0 revealed a less stable structure. In a manner similar to that of known helix bundle apolipoproteins, apoA-V(1-146) induced a relatively small enhancement in 8-anilino-1-naphthalenesulfonic acid fluorescence intensity. Quenching studies with single-Trp apoA-V(1-146) variants revealed that a unique site predicted to reside on the nonpolar face of an amphipathic alpha-helix was protected from quenching by KI. Taken together, the data suggest the 146 N-terminal residues of human apoA-V adopt a helix bundle molecular architecture in the absence of lipid and, thus, likely exist as an independently folded structural domain within the context of the intact protein.     

Citraconylation--a simple method for high protein sequence coverage in MALDI-TOF mass spectrometry

Lysine epsilon -amino group reacts with citraconic anhydride forming a derivative, which is stable on terms for trypsin cleavage. This modification changes the spectrum of peptides formed by the trypsin action; as the number of trypsin-sensitive sites is reduced, the peptides with higher molecular mass can survive in the digest. The various studies of proteins by MALDI-TOF mass spectrometry are often complicated by the low sequence coverage of the peptide chain. This paper demonstrates that the modification of proteins by citraconylation before trypsin cleavage represents a simple experimental technique, which allows a significant increase of sequence coverage in MALDI-TOF mass spectrometry. This improvement is caused both by change of trypsin fragmentation pattern and by disturbance of the protein's native tertiary structure.     

Full-length sequence of the cDNA for human erythroid beta-spectrin

Spectrin is the major molecular consituent of the red cell membrane skeleton. We have isolated overlapping human erythroid beta-spectrin cDNA clones and determined 6773 base pairs of contiguous nucleotide sequence. This includes the entire coding sequence of beta-spectrin. The sequence translates into a 2137 amino acid, 246-kDa peptide. beta-Spectrin is found to consist of three distinct domains. Domain I, at the N terminus, is a 272-amino acid region lacking resemblance to the spectrin repetitive motif. Sequences in this region exhibit striking sequence homology, at both nucleotide and amino acid levels, to the N-terminal "actin-binding" domains of alpha-actinin and dystrophin. Between residues 51 and 270 there is 55% amino acid identity to human dystrophin, with only four single amino acid gaps in alignment. Domain II consists of 17 spectrin repeats. Several sequence variations are observed in typical repeat structure. Homology to alpha-actinin extends beyond domain I into the N-terminal portion of domain II. Domain III, 52 amino acid residues at the C terminus, does not adhere to the spectrin repeat motif. Combining knowledge of spectrin primary structure with previously reported functional studies, it is possible to make several inferences regarding structure/function relationships within the beta-spectrin molecule.     

Changes in structural dynamics of the Grb2 adaptor protein upon binding of phosphotyrosine ligand to its SH2 domain

Growth factor receptor-bound protein 2 (Grb2) is an extensively studied adaptor protein involved in cell signaling. Grb2 is a highly flexible protein composed of a single SH2 domain flanked by two SH3 domains. Here we report on the structural dynamic effects upon interaction of a phosphopeptide ligand derived from the recognition sequence of the Shc adaptor protein with (i) the isolated SH2 domain of Grb2 (Grb2 SH2) and (ii) the full-length Grb2 protein. From kinetic studies using surface plasmon resonance, it was deduced that a conformation change occurred in the SH2 protein as well as the full-length Grb2 after binding. Measurements of hydrogen/deuterium exchange (HDX) in the isolated SH2 domain and full-length Grb2 protein as monitored by electrospray mass spectrometry, showed that binding reduces the overall flexibility of the proteins, possibly via slightly different mechanisms for the single SH2 domain and the full-length Grb2 protein.     

The complete cDNA sequence of bovine coagulation factor V.

Lack of availability of a primary structure for bovine factor V has hindered detailed analysis of a vast majority of structure-function correlations on this molecule. To determine the primary structure of bovine factor V, we used liver mRNA as a template for the synthesis of three cDNA libraries. The sequences of seven overlapping cDNA clones infer two bovine factor V variants. Variant 1 results in a 6910-basepair (bp) cDNA including 103 bp of 5'-untranslated sequence, 6633 bp of coding sequence and 171 bp of 3'-untranslated sequence with a putative polyadenylation site. Variant 2 differs only in the size of the coding sequence (6618 bp). The open reading frame translates to factor V consisting of 2211 (or 2206) amino acids including a 28-amino acid signal peptide. Comparison of the amino acid sequences with human factor Va reveals 84% identity for the heavy and 86% for the light chains. In contrast, the B domain (connecting region) exhibits only 59% identity relative to the human molecule. The bovine B domain contains two repeats of a 14-amino acid structure that is contained only once in the human sequence. Bovine factor V lacks one of the nine amino acid repeats and one of the 17 amino acid repeats present in the human B domain. Factor V has little homology to the factor VIII molecule in the B domain. The 17-amino acid repeat missing in bovine factor V allows identification of an 18-amino acid sequence that is homologous to the B domain of human factor VIII. These 18 amino acids may either constitute the unique vestige of a divergent evolution between the B domains of factors V and VIII or reveal the convergent evolution toward a critical epitope involved in the activation of both procofactors.