A Crystal Structure of the Dengue Virus Non-structural Protein 5 (NS5) Polymerase Delineates Interdomain Amino Acid Residues That Enhance Its Thermostability and de Novo Initiation Activities

The dengue virus (DENV) non-structural protein 5 (NS5) comprises an N-terminal methyltransferase and a C-terminal RNA-dependent RNA polymerase (RdRp) domain. Both enzymatic activities form attractive targets for antiviral development. Available crystal structures of NS5 fragments indicate that residues 263–271 (using the DENV serotype 3 numbering) located between the two globular domains of NS5 could be flexible. We observed that the addition of linker residues to the N-terminal end of the DENV RdRp core domain stabilizes DENV1–4 proteins and improves their de novo polymerase initiation activities by enhancing the turnover of the RNA and NTP substrates. Mutation studies of linker residues also indicate their importance for viral replication. We report the structure at 2.6-Å resolution of an RdRp fragment from DENV3 spanning residues 265–900 that has enhanced catalytic properties compared with the RdRp fragment (residues 272–900) reported previously. This new orthorhombic crystal form (space group P2₁2₁2) comprises two polymerases molecules arranged as a dimer around a non-crystallographic dyad. The enzyme adopts a closed “preinitiation” conformation similar to the one that was captured previously in space group C222₁ with one molecule per asymmetric unit. The structure reveals that residues 269–271 interact with the RdRp domain and suggests that residues 263–268 of the NS5 protein from DENV3 are the major contributors to the flexibility between its methyltransferase and RdRp domains. Together, these results should inform the screening and development of antiviral inhibitors directed against the DENV RdRp.     

The heavy chain of conventional kinesin interacts with the SNARE proteins SNAP25 and SNAP23

Recent studies on the conventional motor protein kinesin have identified a putative cargo-binding domain (residues 827-906) within the heavy chain. To identify possible cargo proteins which bind to this kinesin domain, we employed a yeast two-hybrid assay. A human brain cDNA library was screened, using as bait residues 814-963 of human ubiquitous kinesin heavy chain. This screen initially identified synaptosome-associated protein of 25 kDa (SNAP25) as a kinesin-binding protein. Subsequently, synaptosome-associated protein of 23 kDa (SNAP23), the nonneuronal homologue of SNAP25, was also confirmed to interact with kinesin. The sites of interaction, determined from in vivo and in vitro assays, are the N-terminus of SNAP25 (residues 1-84) and the cargo-binding domain of kinesin heavy chain (residues 814-907). Both regions are composed almost entirely of heptad repeats, suggesting the interaction between heavy chain and SNAP25 is that of a coiled-coil. The observation that SNAP23 also binds to residues 814-907 of heavy chain would indicate that the minimal kinesin-binding domain of SNAP23 and SNAP25 is most likely residues 45-84 (SNAP25 numbering), a heptad-repeat region in both proteins. The major binding site for kinesin light chain in kinesin heavy chain was mapped to residues 789-813 at the C-terminal end of the heavy chain stalk domain. Weak binding of light chain was also detected at the N-terminus of the heavy chain tail domain (residues 814-854). In support of separate binding sites on heavy chain for light chain and SNAPs, a complex of heavy and light chains was observed to interact with SNAP25 and SNAP23.     

Bifunctional structure of two adenylyl cyclases from the myxobacterium Stigmatella aurantiaca

Two adenylyl cyclase genes (cyaA and cyaB) from the myxobacterium Stigmatella aurantiaca were cloned by complementation of Escherichia coli mutants defective in the cya gene. cyaA codes for a protein of 424 amino acid residues (AC1), while cyaB encodes a protein of 352 residues (AC2). Both cyclases are sensitive to adenosine: cAMP production was strongly inhibited in E coli cells and cell extracts expressing these genes. AC1 comprises a hydrophobic domain of six transmembrane helices coupled to a cytoplasmic catalytic domain endowed with adenylyl cyclase activity. A 17 amino acid residue sequence, which is a signature of G-protein coupled receptors, as well as of slime mold Dictyostelium discoideum cyclic AMP receptors, was found in the membrane domain. AC2 displays features also indicating that it is a bifunctional enzyme. The domain located upstream from the catalytic adenylyl cyclase domain shows strong similarity to receiver modules of response regulators of two-component bacterial signaling systems. In vitro mutagenesis of conserved aspartate residues in this domain was shown to interfere with cAMP synthesis.     

Docking and molecular dynamics simulations of the Fyn‐SH3 domain with free and phospholipid bilayer‐associated 18.5‐kDa myelin basic protein (MBP)—Insights into a noncanonical and fuzzy interaction

The molecular details of the association between the human Fyn‐SH3 domain, and the fragment of 18.5‐kDa myelin basic protein (MBP) spanning residues S38–S107 (denoted as xα2‐peptide, murine sequence numbering), were studied in silico via docking and molecular dynamics over 50‐ns trajectories. The results show that interaction between the two proteins is energetically favorable and heavily dependent on the MBP proline‐rich region (P93‐P98) in both aqueous and membrane environments. In aqueous conditions, the xα2‐peptide/Fyn‐SH3 complex adopts a “sandwich”"‐like structure. In the membrane context, the xα2‐peptide interacts with the Fyn‐SH3 domain via the proline‐rich region and the β‐sheets of Fyn‐SH3, with the latter wrapping around the proline‐rich region in a form of a clip. Moreover, the simulations corroborate prior experimental evidence of the importance of upstream segments beyond the canonical SH3‐ligand. This study thus provides a more‐detailed glimpse into the context‐dependent interaction dynamics and importance of the β‐sheets in Fyn‐SH3 and proline‐rich region of MBP. Proteins 2017; 85:1336–1350. © 2017 Wiley Periodicals, Inc.     

Crystal Structure of Active Site-Inhibited Human Coagulation Factor VIIa (des-Gla)

Factor VIIa (FVIIa) is a crucial haemostatic protease consisting of four distinct domains termed the Gla, epidermal growth factor-1 (EGF-1), EGF-2, and protease domains (from N- to C-terminus). The crystal structure of human FVIIa inhibited at the active site with 1, 5-dansyl-Glu-Gly-Arg-chloromethyl ketone and lacking the Gla domain has been solved to a resolution of 2.28 A. The EGF-2 and protease domains were well resolved, whereas no electron density for the EGF-1 domain was observed, suggesting a flexible arrangement or disorder within the crystal. Superposition of the protease domain of the present structure with that previously resolved in the tissue factor (TF)/FVIIai complex revealed that although overall the domain structures are similar, the EGF-2 domain is rotated by 7.5 degrees relative to the protease domain on binding TF. A single cleavage in the protease domain was found, between Arg315 and Lys316 (chymotrypsin numbering 170C-170D) in a FVII-specific insertion loop: this cleavage appeared to be essential for crystallisation. Insertion of the heavy chain N-terminal Ile153 is essentially identical in the two structures, as is the geometry of the active site residues and the inhibitor C-terminal arginine residue. Some differences are seen in the cleaved loop, but changes in TF-contact residues are generally minor. This structure supports the hypothesis that TF binding enables spatial domain arrangements in the flexible FVIIa molecule necessary for procoagulant function and furthermore that active site occupancy induces FVIIa active conformation via N-terminal insertion.     

A proline-rich chitinase from Beta vulgaris

A gene (Chl) encoding a novel type of chitinase was isolated from Beta vulgaris. The Ch1 protein consists of an N-terminal hydrophobic prepeptide of 25 amino acids followed by a hevein-like domain of 22 amino acid residues, an unusually long proline-rich domain of 131 amino acid residues with 90 prolines, and finally a catalytic domain of 261 amino acid residues. Proteins with similar proline-rich domains are present in some other plants. The Chl gene shows a transient expression in response to fungal infection.     

Four hydrophobic amino acid residues in the C-terminal effector domain of the yeast Mig1p repressor are important for its in vivo activity

The Mig1 repressor is a zinc finger protein that mediates glucose repression in yeast. Previous work in Saccharomyces cerevisiae has shown that two domains in Mig1p are required for repression: the N-terminal zinc finger region and a C-terminal effector domain. Both domains are also conserved in Mig1p homologs from the distantly related yeasts Kluyveromyces lactis and K. marxianus, and these Mig1 proteins can fully replace the endogenous Mig1p in S. cerevisiae. We have now made a detailed analysis of the conserved C-terminal effector domain in Mig1p from K. marxianus, using expression in S. cerevisiae to monitor its function. First, a series of small deletions were made within the effector domain. Second, an alanine scan mutagenesis was carried out across the effector domain. Third, double, triple and quadruple mutants were made that affect certain residues within the effector domain. Our results show that four conserved residues within the effector domain, three leucines and one isoleucine, are particularly important for its function in vivo. The analysis further revealed that while the C-terminal effector domain of KmMig1p mediates a seven- to nine-fold repression of the reporter gene, a five- to sixfold residual effect also exists that is independent of the C-terminal effector domain. Similar results were obtained when the corresponding mutations were made in ScMig1p. Moreover, we found that mutations in these residues affect the interaction between Mig1p and the general corepressor subunit Cyc8p (Ssn6p). Modeling of the C-terminal effector domain using a protein of known structure suggests that it may be folded into an α-helix. Received: 30 March 1998 / Accepted: 18 August 1998     

The Last Twenty Residues in the Head Domain of Mouse Lamin A Contain Important Structural Elements for Formation of Head-to-Tail Polymers in Vitro

Nuclear lamins are a type of intermediate filament (IF) proteins. They have a characteristic tripartite domain structure with a α-helical rod domain flanked by non-α-helical N-terminal head and C-terminal tail domains. While the head domain has been shown to be important for the formation of head-to-tail polymers that are critical assembly intermediates for lamin IFs, essential structural elements in this domain have remained obscure. As a first step to remedy this, a series of mouse lamin A mutants in which the head domain (30 amino acid residues) was deleted stepwise from the N-terminus at intervals of 10 residues were bacterially expressed. The assembly properties in vitro of the purified recombinant proteins were explored by electron microscopy. We observed that while a lamin A mutant lacking N-terminal 10 residues formed head-to-tail polymers, a mutant lacking N-terminal 20 residues or the whole head domain (30 residues) showed significantly decreased potency to form head-to-tail polymers. These results suggest that the last 20 residues (from Arg-11 to Gln-30) of the head domain of mouse lamin A contain essential structures for the formation of head-to-tail polymers. The last 20 residues of the head domain include several conserved residues between A- and B-type lamins and also the phosphorylation site for cdc2 kinase, which affects lamin IF organization in vivo and in vitro. Our results provide clues to the molecular mechanism by which the head domain plays a crucial role in lamin polymerization.     

Aligning,analyzing, and visualizing sequences for antibody engineering: Automated recognition of immunoglobulin variable region features

The analysis and comparison of large numbers of immunoglobulin (Ig) sequences that arise during an antibody selection campaign can be time‐consuming and tedious. Typically, the identification and annotation of framework as well as complementarity‐determining regions (CDRs) is based on multiple sequence alignments using standardized numbering schemes, which allow identification of equivalent residues among different family members but often necessitate expert knowledge and manual intervention. Moreover, due to the enormous length variability of some CDRs the benefit of conventional Ig numbering schemes is limited and the calculation of correct sequence alignments can become challenging. Whereas, in principle, a well established set of rules permits the assignment of CDRs from the amino acid sequence alone, no currently available sequence alignment editor provides an algorithm to annotate new Ig sequences accordingly. Here we present a unique pattern matching method implemented into our recently developed ANTIC ALIgN editor that automatically identifies all hypervariable and framework regions in experimentally elucidated antibody sequences using so‐called “regular expressions.” By combination of this widely supported software syntax with the unique capabilities of real‐time aligning, editing and analyzing extended sets of amino acid and/or nucleotide sequences simultaneously on a local workstation, ANTIC ALIgN provides a powerful utility for antibody engineering. Proteins 2016; 85:65–71. © 2016 Wiley Periodicals, Inc.     

The amino acid sequence of a human immunoglobulin G3m(g) pFc' fragment.

Amino acid sequence analysis of the pFc' fragment obtained by pepsin digestion of an IgG3; G3m(g) human myeloma protein HER shows it to consist of 112 residues. It starts at position 334 (gamma1 numbering), contains eight residues of the Cgamma2 region, and the whole Cgamma3 domain. Comparison with the sequence of gamma1 shows five differences including an extra Met at 397. Each is accountable by a single base substitution. The sequence is identical to that of a G3m(b0) molecule except for the previously noted allotype related Tyr/Phe exchange at position 436. The high degree of homology (95%) among gamma-chain subclasses suggests a recent diversification.     

Characteristics of short-chain alcohol dehydrogenases and related enzymes

Different short-chain dehydrogenases are distantly related, constituting a protein family now known from at least 20 separate enzymes characterized, but with extensive differences, especially in the C-terminal third of their sequences. Many of the first known members were prokaryotic, but recent additions include mammalian enzymes from placenta, liver and other tissues, including 15-hydroxyprostaglandin, 17 beta-hydroxysteroid and 11 beta-hydroxysteroid dehydrogenases. In addition, species variants, isozyme-like multiplicities and mutants have been reported for several of the structures. Alignments of the different enzymes reveal large homologous parts, with clustered similarities indicating regions of special functional/structural importance. Several of these derive from relationships within a common type of coenzyme-binding domain, but central-chain patterns of similarity go beyond this domain. Total residue identities between enzyme pairs are typically around 25%, but single forms deviate more or less (14-58%). Only six of the 250-odd residues are strictly conserved and seven more are conserved in all but single cases. Over one third of the conserved residues are glycine, showing the importance of conformational and spatial restrictions. Secondary structure predictions, residue distributions and hydrophilicity profiles outline a common, N-terminal coenzyme-binding domain similar to that of other dehydrogenases, and a C-terminal domain with unique segments and presumably individual functions in each case. Strictly conserved residues of possible functional interest are limited, essentially only three polar residues. Asp64, Tyr152 and Lys156 (in the numbering of Drosophila alcohol dehydrogenase), but no histidine or cysteine residue like in the completely different, classical medium-chain alcohol dehydrogenase family. Asp64 is in the suggested coenzyme-binding domain, whereas Tyr152 and Lys156 are close to the center of the protein chain, at a putative inter-domain, active-site segment. Consequently, the overall comparisons suggest the possibility of related mechanisms and domain properties for different members of the short-chain family.     

A type-1 metacaspase from Acanthamoeba castellanii

The complete sequence of a type-1 metacaspase from Acanthamoeba castellanii is reported comprising 478 amino acids. The metacaspase was recovered from an expression library using sera specific for membrane components implicated in stimulating encystation. A central domain of 155 amino acid residues contains the Cys/His catalytic dyad and is the most conserved region containing at least 30 amino acid identities in all metacaspases. The Acanthamoeba castellanii metacaspase has the most proline-rich N-terminus so far reported in type-1 metacaspases with over 40 prolines in the first 150 residues. Ala–Pro–Pro is present 11 times. Phylogenies constructed using only the conserved proteolytic domains or the complete sequences show identical branching patterns, differing only in the rates of change.     

Comparison of CH1 Domains in Different Classes of Murine Antibodies

The C_H1 domains of antibodies belonging to the following five murine immunoglobulin (Ig) classes IgG1, IgG2a, IgG2b, IgG3 and IgA have been compared. The IgG C_H1 domain structures are, as would be expected, similar overall, but show local conformational variations. When compared with IgG C_H1 domain structures, the IgA C_H1 domain displays several significant structural differences, which are a consequence of insertions/ deletions and specific structural constraints. In regions of structural differences in the IgG C_H1 domains, the spatial correspondence of residues is not reflected by conventional (Kabat) sequence number. Thus the sequence alignment and numbering for C_H1 domains has been revised to be consistent with the three-dimensional alignments.     

Investigation of protonatable residues in Rhodothermus marinus caa 3 haem-copper oxygen reductase: comparison with Paracoccus denitrificans aa 3 haem-copper oxygen reductase

The Rhodothermus marinus caa ₃ haem-copper oxygen reductase contains all the residues of the so-called D- and K-proton channels, with the notable exception of the helix VI glutamate residue (Glu278^I in Paracoccus denitrificans aa ₃), being nevertheless a true oxygen reductase reducing O₂ to water, and an efficient proton pump. Instead, in the same helix, but one turn below, it has a tyrosine residue (Tyr256^I, R. marinus caa ₃ numbering), whose hydroxyl group occupies the same spatial position as the carboxylate group of Glu278^I, as deduced by comparative modelling techniques. Therefore, we proposed previously that this tyrosine residue could play an important role in the proton pathway. In this article we further study this hypothesis, by investigating the equilibrium thermodynamics of protonation in R. marinus caa ₃, using theoretical methodologies based on the structural model previously obtained. Control calculations are also performed for the P. denitrificans aa ₃ oxygen reductase. In both oxygen reductases we find several residues that are proton active (i.e., that display partial protonation) at physiological pH, some of them being redox sensitive (i.e., sensitive to the protein redox state). However, the caa ₃ Tyr256^I is not proton active at physiological pH, in contrast to the aa ₃ Glu278^I which is both proton active at physiological pH and shows a high redox sensitivity. In R. marinus caa ₃ we do not find any other residues in the same protein zone that can have this property. Therefore, there are no putative D-channel residues that are proton active in this oxidase. The protonatable residues of the K-channel are much more functionally conserved in both oxygen reductases than the same type of residues in the D-channel. Two (Tyr262^I and Lys336^I, caa ₃ numbering) out of three protonatable K-channel residues are proton active and redox sensitive in both proteins.     

Human pantothenate kinase 4 is a pseudo‐pantothenate kinase

Pantothenate kinase generates 4′‐phosphopantothenate in the first and rate‐determining step of coenzyme A (CoA) biosynthesis. The human genome encodes three well‐characterized and nearly identical pantothenate kinases (PANK1‐3) plus a putative bifunctional protein (PANK4) with a predicted amino‐terminal pantothenate kinase domain fused to a carboxy‐terminal phosphatase domain. Structural and phylogenetic analyses show that all active, characterized PANKs contain the key catalytic residues Glu138 and Arg207 (HsPANK3 numbering). However, all amniote PANK4s, including human PANK4, encode Glu138Val and Arg207Trp substitutions which are predicted to inactivate kinase activity. Biochemical analysis corroborates bioinformatic predictions—human PANK4 lacks pantothenate kinase activity. Introducing Glu138Val and Arg207Trp substitutions to the human PANK3 and plant PANK4 abolished their robust pantothenate kinase activity. Introducing both catalytic residues back into human PANK4 restored kinase activity, but only to a low level. This result suggests that epistatic changes to the rest of the protein already reduced the kinase activity prior to mutation of the catalytic residues in the course of evolution. The PANK4 from frog, an anamniote living relative encoding the catalytically active residues, had only a low level of kinase activity, supporting the view that HsPANK4 had reduced kinase activity prior to the catalytic residue substitutions in amniotes. Together, our data show that human PANK4 is a pseudo‐pantothenate kinase—a catalytically deficient variant of the catalytically active PANK4 found in plants and fungi. The Glu138Val and Arg207Trp substitutions in amniotes (HsPANK3 numbering) completely deactivated the pantothenate kinase activity that had already been reduced by prior epistatic mutations.     

The amino acid sequence of a protein from wheat kernel closely related to proteins involved in the mechanisms of plant defence

The amino acid sequence of wheatwin1, a monomeric protein of 125 residues isolated from wheat kernel (variety S. Pastore), is reported. Wheatwin1 is highly homologous (95%) to barwin, a protein from barley seed, which was shown to be related to the C-terminal domain of two proteins encoded by the wound-induced geneswin1 andwin2 in potato and to a protein encoded by the same domain of the hevein gene (hev1) in rubber tree. Similarly to barwin, wheatwin1 contains six cysteine residues all linked in disulfide bridges and the N-terminal residue is pyroglutamate. Moreover, structural studies performed on wheatwin1 andwin1 protein by predictive methods demonstrated that these proteins and barwin are closely related in the secondary structure also. The high level of homology found with the product ofwin1,win2, andhev1 genes strongly suggests that barwin and wheatwin1 play a common role in the mechanism of plant defence.     

Fast prediction of protein domain boundaries using conserved local patterns

We have found certain conserved motifs and secondary structural patterns present in the vicinity of interior domain boundary points (dbps) by a data-driven approach without any a priori constraint on the type and number of such features, and without any requirement of sequence homology. We have used these motifs and patterns to rerank the solutions obtained by the well-known domain guess by size (DGS) algorithm. We predict, overall, five solutions. The average accuracy of overall (i.e., top five) predictions by our method [domain boundary prediction using conserved patterns (DPCP)] has improved the average accuracy of the top five solutions of DGS from 71.74 to 82.88 %, in the case of two-continuous-domain proteins, and from 21.38 to 80.56 %, for two-discontinuous-domain proteins. Considering only the top solution, the gains in accuracy are from 0 to 72.74 % for two-continuous-domain proteins with chain lengths up to 300 residues, and from 0 to 62.85 % for those with up to 400 residues. In the case of discontinuous domains, top_min solutions (the minimum number of solutions required for predicting all dbps of a protein) of DPCP improve the average accuracy of DGS prediction from 12.5 to 76.3 % in proteins with chain lengths up to 300 residues, and from 13.33 to 70.84 % for proteins with up to 400 residues. In our validation experiments, the performance of DPCP was also found to be superior to that of domain identification from secondary structure element alignment (DomSSEA), the best method reported so far for efficient prediction of domain boundaries using predicted secondary structure. The average accuracies of the topmost solution of DomSSEA are 61 and 52 % for proteins with up to 300 residues and 400, respectively, in the case of continuous domains; the corresponding accuracies for the discontinuous case are 28 and 21 %.     

Cytotoxicity of the Vibrio vulnificus MARTX toxin Effector DUF5 is linked to the C2A Subdomain

The multifunctional‐autoprocessing repeats‐in‐toxin (MARTX) toxins are bacterial protein toxins that serve as delivery platforms for cytotoxic effector domains. The domain of unknown function in position 5 (DUF5) effector domain is present in at least six different species' MARTX toxins and as a hypothetical protein in Photorhabdus spp. Its presence increases the potency of the Vibrio vulnificus MARTX toxin in mouse virulence studies, indicating DUF5 directly contributes to pathogenesis. In this work, DUF5 is shown to be cytotoxic when transiently expressed in HeLa cells. DUF5 localized to the plasma membrane dependent upon its C1 domain and the cells become rounded dependent upon its C2 domain. Both full‐length DUF5 and the C2 domain caused growth inhibition when expressed in Saccharomyces cerevisiae. A structural model of DUF5 was generated based on the structure of Pasteurella multocida toxin facilitating localization of the cytotoxic activity to a 186 amino acid subdomain termed C2A. Within this subdomain, an alanine scanning mutagenesis revealed aspartate‐3721 and arginine‐3841 as residues critical for cytotoxicity. These residues were also essential for HeLa cell intoxication when purified DUF5 fused to anthrax toxin lethal factor was delivered cytosolically. Thermal shift experiments indicated that these conserved residues are important to maintain protein structure, rather than for catalysis. The Aeromonas hydrophila MARTX toxin DUF5_Ah domain was also cytotoxic, while the weakly conserved C1–C2 domains from P. multocida toxin were not. Overall, this study is the first demonstration that DUF5 as found in MARTX toxins has cytotoxic activity that depends on conserved residues in the C2A subdomain. Proteins 2014; 82:2643–2656. © 2014 Wiley Periodicals, Inc.     

Structure of actinidin: details of the polypeptide chain conformation and active site from an electron density map at 2-8 A resolution

A 2·8 Å resolution electron density map of the sulphydryl protease, actinidin, has been calculated. Two isomorphous heavy-atom derivatives, prepared with uranyl acetate and dichloroethylenediamineplatinum(II), were used to calculate phases by the method of isomorphous replacement, giving an overall figure of merit of 0·81. The polypeptide chain is well-defined in the present map and many side-chains can be identified from their appearance. The molecule consists of a single chain of 220 residues, the last two of which appear disordered in the map and contains at least two, and probably three, disulphide bridges. The conformation of the polypeptide chain is remarkably similar to that of papain. It is folded into two domains, domain I consisting of residues 19–115 and 214–218, and domain II residues 1–18 and 116–213. There are three significant stretches of α-helix, involving residues 25–42, 69–81 and 120–129, together with several shorter pieces, while much of domain II consists of a twisted β-sheet structure. When compared with papain, actinidin has two additional residues inserted between 59 and 60, one inserted between 78 and 79, and four between 168 and 169 (papain numbering) while one residue (194) has been deleted from the papain structure. All these changes are in external parts of the molecule and have little effect on the conformation. The positions and orientations of the catalytically-important side-chains in the active site are virtually identical with those in papain, but some of the side-chains lining the non-polar binding pocket are clearly different.     

Localization of the heparin binding exosite of factor IXa

Factor IXa (FIXa) is known to have a binding site for heparin that has not been mapped by a mutagenesis study. By homology modeling based on structural data, we identified eight basic residues in the catalytic domain of FIXa that can potentially bind to heparin. These residues, Lys(98), Lys(126), Arg(165), Arg(170), Lys(173), Lys(230), Arg(233), and Lys(239) (chymotrypsin numbering) were substituted with Ala in separate constructs in Gla-domainless forms. Following activation, it was found that all FIXa derivatives cleaved the chromogenic substrate CBS 31.39 with near normal catalytic efficiencies. Similarly, antithrombin inactivated FIXa derivatives with a similar second-order association rate constant (k(2)) in both the absence and presence of pentasaccharide. In the presence of a full-length heparin, however, k(2) values were dramatically impaired with certain mutants. Direct binding studies revealed that the same mutants lost their affinities for binding to heparin-Sepharose. Both kinetic and direct binding data indicated that five basic residues of FIXa in the following order of importance, Arg(233) > Arg(165) > Lys(230) > Lys(126) > Arg(170) are critical for binding to heparin. Consistent with these results, examination of the crystal structure of the catalytic domain of FIXa indicated that all five basic residues are spatially aligned in a manner optimal for interaction with heparin.