Crystallization and preliminary X-ray crystallographic studies of wild-type human ornithine transcarbamylase and two naturally occurring mutants at position 277.

Wild-type human ornithine transcarbamylase (OTCase) and two mutants (R277Q and R277W) that cause 'late-onset' hyperammonemia were crystallized and a preliminary structure determination was carried out. The unliganded wild-type enzyme crystallizes in the cubic space group I23, with unit-cell parameters a = b = c = 203.4 A. R277Q crystallizes in two crystal forms under the same crystallization conditions. One crystal form is isomorphous to that of unliganded wild-type crystals, with unit-cell parameters a = b = c = 202.2 A. The second form also belongs to a cubic space group, P4(3)32, but has unit-cell parameters a = b = c = 139.8 A. R277W crystals are isomorphous to the second crystal form of R277Q, with unit-cell parameters a = b = c = 138.7 A. None of these crystal forms is isomorphous to other crystal forms of OTCase that have been studied. The structures in both crystal forms have been solved using molecular replacement. In the first crystal form there are two monomers in the asymmetric unit, corresponding to a solvent content of 75%. Because of its high molecular and crystal symmetry and the presence of non-crystallographic symmetry, this structure could not be solved with AMoRe or X-PLOR, but was solved successfully with COMO. There is only one monomer in the asymmetric unit in the second crystal form, corresponding to a solvent content of 62%. This structure was successfully solved with AMoRe.

Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes.

Two crystal structures of human ornithine transcarbamylase (OTCase) complexed with the substrate carbamoyl phosphate (CP) have been solved. One structure, whose crystals were prepared by substituting N-phosphonacetyl-L-ornithine (PALO) liganded crystals with CP, has been refined at 2.4 A (1 A=0.1 nm) resolution to a crystallographic R factor of 18.4%. The second structure, whose crystals were prepared by co-crystallization with CP, has been refined at 2.6 A resolution to a crystallographic R factor of 20.2%. These structures provide important new insights into substrate recognition and ligand-induced conformational changes. Comparison of these structures with the structures of OTCase complexed with the bisubstrate analogue PALO or CP and L-norvaline reveals that binding of the first substrate, CP, induces a global conformational change involving relative domain movement, whereas the binding of the second substrate brings the flexible SMG loop, which is equivalent to the 240s loop in aspartate transcarbamylase, into the active site. The model reveals structural features that define the substrate specificity of the enzyme and that regulate the order of binding and release of products.

The clinically variable R40H mutant ornithine carbamoyltransferase shows cytosolic degradation of the precursor protein in CHO cells.

Ornithine carbamoyltransferase (OCT) deficiency is now frequently found in adults with hyperammonaemia affected by mutations that cause partial deficiency of this urea cycle enzyme. One of these mutations (R40H) has occurred in several families and has been found also in asymptomatic relatives. To better understand the phenotypic heterogeneity of this recurrent mutation, we investigated the biological properties of the mutant protein. Using 35S labelling, the import and processing of the R40H mutant OCT protein was investigated in intact CHO cells and in isolated rat liver mitochondria and compared to the wild type and R141Q mutant that causes complete enzyme deficiency. The R40H OCT protein seems to be imported and processed by the mitochondria in a manner similar to that of wild type. However, it is consistently degraded to a smaller fragment in the intact cells, unlike the wild type and R141Q mutant. The mature form of the enzyme is not susceptible to degradation. These data, obtained in CHO cells, suggest that deficiency in OCT enzymatic function conferred by the R40H mutation is likely caused by enhanced degradation of the preprotein in the cytosol. We propose therefore that variation in the rate of OCT turnover is responsible for the heterogeneity of the clinical phenotype in these patients.

Crystal structure of human ornithine transcarbamylase complexed with carbamoyl phosphate and L-norvaline at 1.9 A resolution.

The crystal structure of human ornithine transcarbamylase (OTCase) complexed with carbamoyl phosphate (CP) and L-norvaline (NOR) has been determined to 1.9-A resolution. There are significant differences in the interactions of CP with the protein, compared with the interactions of the CP moiety of the bisubstrate analogue N-(phosphonoacetyl)-L-ornithine (PALO). The carbonyl plane of CP rotates about 60 degrees compared with the equivalent plane in PALO complexed with OTCase. This positions the side chain of NOR optimally to interact with the carbonyl carbon of CP. The mixed-anhydride oxygen of CP, which is analogous to the methylene group in PALO, interacts with the guanidinium group of Arg-92; the primary carbamoyl nitrogen interacts with the main-chain carbonyl oxygens of Cys-303 and Leu-304, the side chain carbonyl oxygen of Gln-171, and the side chain of Arg-330. The residues that interact with NOR are similar to the residues that interact with the ornithine (ORN) moiety of PALO. The side chain of NOR is well defined and close to the side chain of Cys-303 with the side chains of Leu-163, Leu-200, Met-268, and Pro-305 forming a hydrophobic wall. C-delta of NOR is close to the carbonyl oxygen of Leu-304 (3.56 A), S-gamma atom of Cys-303 (4.19 A), and carbonyl carbon of CP (3.28 A). Even though the N-epsilon atom of ornithine is absent in this structure, the side chain of NOR is positioned to enable the N-epsilon of ornithine to donate a hydrogen to the S-gamma atom of Cys-303 along the reaction pathway. Binding of CP and NOR promotes domain closure to the same degree as PALO, and the active site structure of CP-NOR-enzyme complex is similar to that of the PALO-enzyme complex. The structures of the active sites in the complexes of aspartate transcarbamylase (ATCase) with various substrates or inhibitors are similar to this OTCase structure, consistent with their common evolutionary origin.

Crystal structure of bullfrog M ferritin at 2.8 A resolution: analysis of subunit interactions and the binuclear metal center.

Ferritins concentrate and store iron as a mineral in all bacterial, plant, and animal cells. The two ferritin subunit types, H or M (fast) and L (slow), differ in rates of iron uptake and mineralization and assemble in vivo to form heteropolymeric protein shells made up of 24 subunits; H/L subunit ratios reflect cell specificity of H and L subunit gene expression. A diferric peroxo species that is the initial reaction product of Fe(II) in H-type ferritins, as well as in ribonucleotide reductase (R2) and methane monooxygenase hydroxylase (MMOH), has recently been characterized, exploiting the relatively high accumulation of the peroxo intermediate in frog H-subunit type recombinant ferritin with the M sequence. The stability of the diferric reaction centers in R2 and MMOH contrasts with the instability of diferric centers in ferritin, which are precursors of the ferric mineral. We have determined the crystal structure of the homopolymer of recombinant frog M ferritin in two crystal forms: P4(1)2(1)2, a = b = 170.0 A and c = 481.5 A; and P3(1)21, a = b = 210.8 A and c = 328.1 A. The structural model for the trigonal form was refined to a crystallographic R value of 19.0% (Rfree = 19.4%); the two structures have an r.m.s.d. of approximately 0.22 A for all C alpha atoms. Comparison with the previously determined crystal structure of frog L ferritin indicates that the subunit interface at the molecular twofold axes is most variable, which may relate to the presence of the ferroxidase site in H-type ferritin subunits. Two metal ions (Mg) from the crystallization buffer were found in the ferroxidase site of the M ferritin crystals and interact with Glu23, Glu58, His61, Glu103, Gln137 and, unique to the M subunit, Asp140. The data suggest that Gln137 and Asp140 are a vestige of the second GluxxHis site, resulting from single nucleotide mutations of Glu and His codons and giving rise to Ala140 or Ser140 present in other eukaryotic H-type ferritins, by additional single nucleotide mutations. The observation of the Gln137xxAsp140 site in the frog M ferritin accounts for both the instability of the diferric oxy complexes in ferritin compared to MMOH and R2 and the observed kinetic variability of the diferric peroxo species in different H-type ferritin sequences.

The magnitude of the allosteric conformational transition of aspartate transcarbamylase is altered by mutations.

Global conformational transitions are of central functional importance for many enzymes and binding proteins. It is not known, however, how much variability can exist in such structural-functional linkages. We have characterized the global magnitude of the T to R conformational transition of Escherichia coli aspartate transcarbamylase (ATCase) by measuring (1) hydration changes using osmotic stress and (2) hydrodynamic changes using high-precision analytical gel chromatography. We find that specific mutations can alter the structural magnitude of the enzyme's conformational transition without abolishing allostery, suggesting that some degree of plasticity exists in the conformational component of allostery.

1.85-A resolution crystal structure of human ornithine transcarbamoylase complexed with N-phosphonacetyl-L-ornithine. Catalytic mechanism and correlation with inherited deficiency.

The crystal structure of human ornithine transcarbamoylase complexed with the bisubstrate analog N-phosphonacetyl-L-ornithine has been solved at 1.85-A resolution by molecular replacement. Deleterious mutations produce clinical hyperammonia that, if untreated, results in neurological symptoms or death (ornithine transcarbamylase deficiency). The holoenzyme is trimeric, and as in other transcarbamoylases, each subunit contains an N-terminal domain that binds carbamoyl phosphate and a C-terminal domain that binds L-ornithine. The active site is located in the cleft between domains and contains additional residues from an adjacent subunit. Binding of N-phosphonacetyl-L-ornithine promotes domain closure. The resolution of the structure enables the role of active site residues in the catalytic mechanism to be critically examined. The side chain of Cys-303 is positioned so as to be able to interact with the delta-amino group of L-ornithine which attacks the carbonyl carbon of carbamoyl phosphate in the enzyme-catalyzed reaction. This sulfhydryl group forms a charge relay system with Asp-263 and the alpha-amino group of L-ornithine, instead of with His-302 and Glu-310, as previously proposed. In common with other ureotelic ornithine transcarbamoylases, the human enzyme lacks a loop of approximately 20 residues between helix H10 and beta-strand B10 which is present in prokaryotic ornithine transcarbamoylases but has a C-terminal extension of 10 residues that interacts with the body of the protein but is exposed. The sequence of this C-terminal extension is homologous to an interhelical loop found in several membrane proteins, including mitochondrial transport proteins, suggesting a possible mode of interaction with the inner mitochondrial membrane.

Intersubunit hydrogen bond acts as a global molecular switch in Escherichia coli aspartate transcarbamoylase.

Tyr 165 in the catalytic subunit of Escherichia coli aspartate transcarbamoylase (ATCase, EC 2.1.3.2) forms an intersubunit hydrogen bond in the T state with Glu 239 in the 240s loop of a second catalytic subunit, which is broken in the T to R transition. Substitution of Tyr 165 by Phe lowers substrate affinity by approximately an order of magnitude and alters the pH profile for enzyme function. We have determined the crystal structure of Y165F at 2.4 A resolution by molecular replacement, using a wild-type T state structure as the probe, and refined it to an R value of 25.2%. The Y165F mutation induces a global conformational change that is in the opposite direction to the T to R transition and therefore results in an extreme T state. The two catalytic trimers move closer by approximately 0.14 A and rotate by approximately 0.2 degrees , in the opposite direction to the T-->R rotation; the two domains of each catalytic chain rotate by approximately 2.1 degrees, also in the opposite direction to the T-->R transition; and the 240s loop adopts a new conformation. Residues 229 to 236 shift by approximately 2.4 A so that the active site is more open. Residues 237 to 244 rotate by approximately 24.1 degrees, altering interactions within the 240s loop and at the C1-C4 and C1-R4 interfaces. Arg 167, a key residue in domain closure and interactions with L-Asp, swings out from the active site to interact with Tyr 197. This crystal structure is consistent with the functional properties of Y165F, expands our knowledge of the conformational repertoire of ATCase, and indicates that the canonical T state does not represent an extreme.

The biochemical and molecular spectrum of ornithine transcarbamylase deficiency.

Ornithine transcarbamylase (OTCase) deficiency, the most common inherited urea cycle disorder, is transmitted as an X-linked trait. The clinical phenotype in affected males as well as heterozygous females shows a spectrum of severity ranging from neonatal hyperammonaemic coma to asymptomatic adults. The ornithine transcarbamylase enzyme is a trimer with three active sites per holoenzyme molecule, each of which is composed of an interdomain region of one polypeptide and a polar domain of the adjacent polypeptide. The OTC gene is located on the short arm of the X-chromosome and one of the two alleles undergoes inactivation in female cells. Approximately 140 mutations have been found in families affected with OTCase deficiency, most having their own 'private' mutation. Large deletions of one exon or more are seen in approximately 7% of patients, small deletions or insertions are seen in about 9%, and the remaining mutations are single base substitutions. Approximately 15% of mutations affect RNA splicing sites. The recurrent mutations are distributed equally among CpG dinucleotide hot spots. Generally, mutations causing neonatal disease affect amino acid residues that are 'buried' in the interior of the enzyme, especially around the active site, while those associated with late onset and milder phenotypes tend to be located on the surface of the protein. Very few mutations have been found in the sequence of the leader peptide, proportionally much fewer than in the sequence of the mature enzyme. Only few of the mutations have been expressed in bacteria or mammalian cells for the study of their deleterious mechanisms. Examples of expressed mutations include R277W and R277Q associated with late-onset disease, which markedly increase the Km for ornithine, shift the pH optimum to more alkaline and decrease the thermal stability of the purified mutant enzyme. R141Q (neonatal disease) disrupts the active site, whereas the purified R40H mutant has normal catalytic function and this mutation is likely to affect posttranslational processing such as mitochondrial targeting. It appears that most new mutations occur in male sperm and are then passed on to a transmitting heterozygous female. Uncommonly, mild mutations are transmitted by asymptomatic males to their daughters, subsequently resulting in clinical disease of males in future generations. The causes for variable expressivity of these mutations are currently unknown but are likely to involve a combination of environmental and genetic modifiers.

Effects of the T-->R transition on the electrostatic properties of E. coli aspartate transcarbamylase.

Aspartate transcarbamylase is a large (310 kD), multisubunit protein that binds substrates cooperatively and undergoes a large change in quaternary structure when substrates bind. The forces that drive this transition are poorly understood. We evaluated the electrostatic component of these forces by using finite difference and multigrid methods to solve the nonlinear Poisson-Boltzmann equation for complexes of the enzyme with several substrates and substrate analogs. The results have been compared with calculations for the unliganded protein. While pK1/2 values of most ionizable residues fall within 3 pH units of values for model compounds, 31 have pK1/2 values that fall outside the range 0-17. Many of these residues are at the active site, where they interact with the highly charged substrate, in the 80s loop or 240s loop or interact with these loops. The pK1/2 values of eight ionizable residues related by the twofold molecular axes differ by more than 3 pH units, providing additional evidence for asymmetry within the crystal. As in the unliganded structure, a set of residues forms a network in which ionizable groups with Wij values greater than 2 kcal-m(-1) are separated by distances greater than 5 A. Some residues participate in this network in both the unliganded and N-phosphonacetyl-L-aspartate (PALA)-liganded structure, while others are found in only one structure. The network is more extensive in the PALA-liganded structure than in the unliganded structure, but consists of two separate networks in the two halves of the molecule.

Localized unfolding at the junction of three ferritin subunits. A mechanism for iron release?

How and where iron exits from ferritin for cellular use is unknown. Twenty-four protein subunits create a cavity in ferritin where iron is concentrated >10(11)-fold as a mineral. Proline substitution for conserved leucine 134 (L134P) allowed normal assembly but increased iron exit rates. X-ray crystallography of H-L134P ferritin revealed localized unfolding at the 3-fold axis, also iron entry sites, consistent with shared use sites for iron exit and entry. The junction of three ferritin subunits appears to be a dynamic aperture with a "shutter" that cytoplasmic factors might open or close to regulate iron release in vivo.

Solvent perturbation of the allosteric regulation of aspartate transcarbamylase.

Escherichia coli aspartate transcarbamylase (ATCase) catalyzes the first committed step in pyrimidine biosynthesis, the condensation of aspartate and carbamyl phosphate. ATCase is positively allosterically regulated by ATP and negatively regulated by CTP. We have used mild solvent perturbation to gain global molecular information about the mechanism of heterotropic allostery. The [NaCl], temperature, and osmotic pressure dependence of the enzymatic activity of ATCase has been examined in the presence and absence of allosteric effectors. The results indicate that: 1) Regulation of aspartate binding by CTP appears to involve a unique set of electrostatic interactions not involved in enzyme function in the presence of ATP or in the absence of effectors. 2) Aspartate binding is enthalpically driven in the presence and absence of allosteric effectors. 3) The apparent enthalpy and entropy of aspartate binding (delta H, delta S), and activation energy of catalysis (Ea) are substantially altered in the presence of CTP but not ATP. 4) The change in hydration of ATCase upon substrate binding is the same in the presence and absence of allosteric effectors. 5) The linkage between heterotropic and homotropic allostery is different for ATP and CTP.

Substrate-induced conformational change in a trimeric ornithine transcarbamoylase.

The crystal structure of Escherichia coli ornithine transcarbamoylase (OTCase, EC 2.1.3.3) complexed with the bisubstrate analog N-(phosphonacetyl)-L-ornithine (PALO) has been determined at 2.8-A resolution. This research on the structure of a transcarbamoylase catalytic trimer with a substrate analog bound provides new insights into the linkages between substrate binding, protein-protein interactions, and conformational change. The structure was solved by molecular replacement with the Pseudomonas aeruginosa catabolic OTCase catalytic trimer (Villeret, V., Tricot, C., Stalon, V. & Dideberg, O. (1995) Proc. Natl. Acad. Sci. USA 92, 10762-10766; Protein Data Bank reference pdb 1otc) as the model and refined to a crystallographic R value of 21.3%. Each polypeptide chain folds into two domains, a carbamoyl phosphate binding domain and an L-ornithine binding domain. The bound inhibitor interacts with the side chains and/or backbone atoms of Lys-53, Ser-55, Thr-56, Arg-57, Thr-58, Arg-106, His-133, Asn-167, Asp-231, Met-236, Leu-274, Arg-319 as well as Gln-82 and Lys-86 from an adjacent chain. Comparison with the unligated P. aeruginosa catabolic OTCase structure indicates that binding of the substrate analog results in closure of the two domains of each chain. As in E. coli aspartate transcarbamoylase, the 240s loop undergoes the largest conformational change upon substrate binding. The clinical implications for human OTCase deficiency are discussed.

Functionally linked hydration changes in Escherichia coli aspartate transcarbamylase and its catalytic subunit.

Aspartate transcarbamylase (ATCase) is a highly regulated, dodecameric enzyme that catalyzes the first committed step in pyrimidine biosynthesis. Upon ligation, ATCase undergoes a conformational transition from a low-activity T-state to a high-activity R-state. This transition involves major changes in the molecular architecture, including structural rearrangements of several intersubunit interfaces and a 12 A expansion of the molecule along its 3-fold axis. Solute-induced osmotic stress experiments report that approximately 208 solvent waters are taken up by ATCase as it binds substrate. Solvent-accessible surface area calculations conducted on the T and R conformers of ATCase agree very well with this result, predicting that approximately 189 waters are taken up during this conformational change. Both osmotic stress measurements and surface area calculations on the catalytic trimer of ATCase predict water release upon ligation of the trimer. Specific aspects of the application of osmotic stress to ATCase are also discussed, including solute size effects, and an assessment of potential alternative explanations for these results.

Identification of 'private' mutations in patients with ornithine transcarbamylase deficiency.

The majority of cases of ornithine transcarbamylase deficiency are due to novel mutations making it impossible to develop common methods for genetic analysis. However, identification of causative mutations has important implications for diagnosis (particularly prenatal diagnosis), prediction of likely course and outcome and the eventual possibility of gene therapy. As part of a continuing study of ornithine transcarbamylase deficiency, we now report an additional thirty novel mutations in the ornithine transcarbamylase gene, together with a brief summary of their clinical presentations.

'Late onset' ornithine transcarbamylase deficiency: function of three purified recombinant mutant enzymes.

Although many mutations in the ornithine transcarbamylase gene have been correlated with 'late onset' of hyperammonemia in patients, the effects of these mutations on enzyme function are largely unknown. Three recurrent mutations (R40H, R277W and R277Q) found in patients with 'late onset' disease were incorporated into 'mature' human ornithine transcarbamylase cDNA and overexpressed in Escherichia coli. The three recombinant mutant enzymes were purified to homogeneity on an affinity column and their biochemical characteristics were compared to the wild type enzyme. The R277W and R277Q mutants display markedly reduced affinity for L-ornithine, loss of substrate inhibition, alkaline shift of pH optimum, and reduced thermal stability compared to the wild type enzyme. These differences, particularly the reduced affinity for L-ornithine, are sufficient to account for their biochemical effects. In contrast, the 'mature' R40H mutant was biochemically indistinguishable from the wild type enzyme in vitro.

Expression, purification and kinetic characterization of wild-type human ornithine transcarbamylase and a recurrent mutant that produces 'late onset' hyperammonaemia.

Ornithine Transcarbamylase Deficiency, an X-linked disorder, is the most common cause of inherited urea cycle disorders. Approx. 90 mutations that produce reduced levels of ornithine transcarbamylase (OTCase) activity have been identified in patients [Tuchman (1993) Hum. Mutat. 2, 174-178; Tuchman and Plante (1995) Hum. Mutat. 5, 293-295]. A model of the three-dimensional structure of OTCase, developed on the basis of its homology to the catalytic subunit of Escherichia coli aspartate transcarbamylase (ATCase) [Tuchman, Morizono, Reish, Yuan and Allewell (1995) J. Med. Genet. 32, 680-688], and in good agreement with the crystal structure of Pseudomonas aeruginosa OTCase [Villeret, Tricot, Stalon and Dideberg (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10762-10766], indicates that many mutations that produce severe clinical symptoms are at the active site or buried in the interior of the protein. However, one of the few recurrent mutations, R277W, an alteration that produces a milder phenotype of ornithine transcarbamylase deficiency, is located in the model in a loop remote from the active site that is analogous to a similar loop (the 240's loop, a flexible loop of the catalytic chain of Escherichia coli aspartate transcarbamylase, comprised of residues 230-250) of ATCase. Human wild-type OTCase and the R277W mutant have been cloned and overexpressed in E. coli and a rapid and efficient purification method utilizing the bisubstrate analogue, Ndelta-(phosphonacetyl)-L-ornithine, has been developed and used to purify both proteins. Gel chromatography indicates both are trimeric. The pH dependence of the kinetic parameters of the wild-type enzyme is similar to that of E. coli OTCase [Kuo, Herzberg and Lipscomb (1985) Biochemistry 24, 4754-4761], suggesting that its catalytic mechanism is similar, although its maximal activity is approx. 10-fold less. Compared with the wild-type, the R277W mutant has nearly 70-fold lower affinity for L-ornithine, shows no substrate inhibition, and its thermal stability is reduced by 5 degrees C. Its reduced affinity for L-ornithine, which in turn results in lower activity at physiological concentrations of ornithine, as well as its reduced stability, may contribute to the clinical effects that it produces.

Is substrate inhibition a consequence of allostery in aspartate transcarbamylase?

Aspartate transcarbamylase (ATCase) is a highly regulated, multisubunit enzyme that catalyzes the first regulated step in pyrimidine biosynthesis. Although ATCase exhibits strong substrate inhibition (the reduction of enzyme activity at high substrate concentrations), the mechanism of substrate inhibition has not been investigated. At the molecular level, substrate inhibition may result either from local events at the active site or from global or specific long-range allosteric effects. We have compared the results of fitting kinetic data to several models: (a) a semi-empirical steady-state kinetic model that includes cooperative substrate binding (described by a Hill coefficient) and partial uncompetitive substrate inhibition, (b) a nested allosteric model developed to analyze substrate inhibition of the ATPase activity of GroEL, an enzyme with a quaternary structure analogous to ATCase (O. Yifrach and A. Horovitz, Biochemistry, 34 (1995) 5303), and (c) purely concerted models, including a model originally proposed by Monod et al. (J. Monod, J. Wyman and J.P. Changeux, J. Mol. Biol., 12 (1965) 88). Model (a) is the first kinetic equation for ATCase that both fits the data and returns physically realistic values for all parameters, but it is a modified Hill equation and thus returns little or no molecular mechanistic information. The nested allosteric model (b), which assumes concerted cooperativity within each catalytic trimer of ATCase and sequential cooperativity between trimers, is unlikely to be the correct model for ATCase, since isolated catalytic trimers, which cannot exhibit the sequential cooperativity of the model, still exhibit substrate inhibition. Analysis of concerted models (c) shows that a two-state model is inadequate to account for substrate inhibition in ATCase. Further, although unique fits to a three-state model cannot be obtained, because the parameters are highly correlated, several sets of parameter values fit the data well and are in accord with other experimental results. These results indicate that substrate inhibition in ATCase may be the consequence of allostery, and that further experimental investigation is warranted.

Ionic properties of membrane association by vitamin K-dependent proteins: the case for univalency.

Ionic properties of membrane interaction by prothrombin, protein Z, and other vitamin K-dependent proteins were studied to determine the relevance of a monovalent membrane contact mechanism between one phospholipid headgroup and a calcium-lined pore in the protein [McDonald, J. F., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsestuen, G. L. (1997) Biochemistry 36, 5120-5127]. For comparison, multivalent ionic interaction was illustrated by peptides of +3 to +5 net charge and by blood clotting factor V. As expected, the peptides were easily dissociated by salt and gave nominal charge-charge interactions (zazb values) of -13 to -17. Factor V showed much higher binding affinity despite nominal zazb values of about 9. Membrane-bound prothrombin and protein Z showed very low sensitivity to salt as long as calcium was at saturating levels (zazb values of approximately -1.3 to -1.4), appropriate for univalent ionic attraction. Prothrombin contains +3 charge groups (Lys-2, Lys-11, Arg-10) that are absent from the GLA domain (residues 1-35) of protein Z, while protein Z contains -4 charge groups (Gla-11, Asp-34, Asp-35) that are absent in prothrombin. Thus, similar zazb relationships indicated little role for these surface charges in direct membrane contact. Calcium-saturated protein Z bound to phosphatidylcholine (PC) in a manner which indicated the addition of one calcium ion, bringing the total calcium stoichiometry in the protein-membrane complex to at least 8. Protein Z bound to phosphatidic acid (PA) in a manner suggesting the need for a fully ionized phosphate headgroup, a property expected by ion pairing in an isolated environment. Electrostatic calculations showed that the proposed protein site for phosphate interaction was electropositive. The cluster of hydrophobic amino acids (Phe-5, Leu-6, and Val-9) on the surface of prothrombin was electronegative, suggesting a role in the electrostatic architecture of the GLA domain. Overall, membrane binding by vitamin K-dependent proteins appeared consistent with the formation of an ion pair in an isolated environment.