A study on the effects of linker flexibility on acid phosphatase PhoC-GFP fusion protein using a novel linker library

Fusion strategy has been widely used to construct artificial multifunction proteins. The flexibility or rigidity of linkers between two fused partners is an important parameter that affects the function of fusion proteins. By combining the flexible unit GGGGS (F) and rigid unit EAAAK (R), ten linkers consisting of five elementary units that cover the fully rigid RRRRR linker to the fully flexible FFFFF linker were used to construct acid phosphatase-green fluorescence protein fusion protein (PhoC-GFP). By varying the linker flexibility in PhoC-GFPs, the relative specific activity of phosphotransferase and phosphatase varied from ∼19.0% to 100% and ∼9.35% to 100%, respectively. There exists an optimal linker capable of achieving the highest phosphotransferase/phosphatase activity and GFP fluorescence intensity. We found that the highest activities were achieved neither with the rigid RRRRR linker nor with the flexible FFFFF linker, but with the FFFRR linker. Linker flexibility could adjust the activity ratio between phosphotransferase and phosphatase and varied between ∼30% to 100%. PhoC-GFP with FRRRR linker achieved the highest relative specific phosphotransferase activity/relative specific phosphatase activity (T/P) value. Our results show that applying a linker library with controllable flexibility to the fusion proteins will be an efficient way to adjust the function of fusion enzymes.     

Probing the primary structural determinants of streptokinase inter-domain linkers by site-specific substitution and deletion mutagenesis

The bacterial protein streptokinase (SK) contains three independently folded domains (α, β and γ), interconnected by two flexible linkers with noticeable sequence homology. To investigate their primary structure requirements, the linkers were swapped amongst themselves i.e. linker 1 (between α and β domains) was swapped with linker 2 (between β and γ domains) and vice versa. The resultant construct exhibited very low activity essentially due to an enhanced proteolytic susceptibility. However, a SK mutant with two linker 1 sequences, which was proteolytically as stable as WT-rSK retained about 10% of the plasminogen activator activity of rSK When the native sequence of each linker was substituted with 9 consecutive glycine sequences, in case of the linker 1 substitution mutant substantial activity was seen to survive, whereas the linker 2 mutant lost nearly all its activity. The optimal length of linkers was then studied through deletion mutagenesis experiments, which showed that deletion beyond three residues in either of the linkers resulted in virtually complete loss of activator activity. The effect of length of the linkers was then also examined by insertion of extraneous pentapeptide sequences having a propensity for adopting either an extended conformation or a relatively rigid conformation. The insertion of poly-Pro sequences into native linker 2 sequence caused up to 10-fold reduction in activity, whereas its effect in linker 1 was relatively minor. Interestingly, most of the linker mutants could form stable 1:1 complexes with human plasminogen. Taken together, these observations suggest that (i) the functioning of the inter-domain linkers of SK requires a critical minimal length, (ii) linker 1 is relatively more tolerant to insertions and sequence alterations, and appears to function primarily as a covalent connector between the α and β domains, and (iii) the native linker 2 sequence is virtually indispensable for the activity of SK probably because of structural and/or flexibility requirements in SK action during catalysis.     

Development of a 3′-amino linker with high conjugation activity and its application to conveniently cross-link blunt ends of a duplex

The 2-aminoethyl carbamate linker (ssH linker) exhibits high activity in modifying the 5′-termini of oligonucleotides; however, the ssH linker is not appropriate for 3′-terminal modification because it undergoes intramolecular trans-acylation under heat–aqueous ammonia conditions. We developed an N-(2-aminoethyl)carbamate linker (revH linker), in which the carbamate is oriented in the reverse direction relative to that in 2-aminoethyl carbamate. The revH linker was tolerant to heat–alkaline conditions and retained its high reactivity in conjugation with exogenous molecules. The 3′-revH linker was efficiently linked with the 5′-ssH linker at the termini of complementary double strands with a bifunctional molecule, producing a synthetic loop structure. An anti-microRNA oligonucleotide (AMO) was prepared from the chemical ligation of three-stranded 2′-O-methyl RNAs, and the AMO with two alkyl loops exhibited high inhibition activity toward miRNA function. The revH linker is not only useful for 3′-terminal modification of oligonucleotides but also expands the utility range in combination with the 5′-ssH linker.     

Nuclear protein modification and chromatin substructure. 2. Internucleosomal localization of poly(adenosine diphosphate-ribose) polymerase.

Definitive evidence for poly(ADP-Rib) polymerase activity is localized within internucleosomal "linker" regions of HeLa cell chromatin is presented. This evidence was based on the following criteria: the enzyme activity did not coincide with the position of core particles in a sucrose gradient but was displaced to that part of the gradient which is enriched in monomers with linker regions. This was not due to dimer contamination, since resedimentation did not affect the enzyme activity in relation to the monomer. A new method of assaying enzyme activity directly in polyacrylamide gels following the separation of monomers and dimers showed that only dimers and monomers with linker regions contained activity. When dimers were digested, the enzyme activity moved from the dimer to the monomer with linker.     

The chaperone function of cyclophilin 40 maps to a cleft between the prolyl isomerase and tetratricopeptide repeat domains

Cyclophilin 40 (CyP40), an immunophilin cochaperone present in steroid receptor-Hsp90 complexes, contains an N-terminal peptidylprolyl isomerase (PPIase) domain separated from a C-terminal Hsp90-binding tetratricopeptide repeat (TPR) domain by a 30-residue linker. To map CyP40 chaperone function, CyP40 deletion mutants were prepared and analysed for chaperone activity. CyP40 fragments containing the PPIase domain plus linker or the linker region and the adjoining TPR domain retained chaperone activity, whilst individually, the catalytic and TPR domains were devoid of chaperoning ability. CyP40 chaperone function then, is localized within the linker that forms a binding cleft with potential to accommodate non-native substrates.     

The "linker" region (amino acids 38-47) of the disintegrin elegantin is a novel inhibitory domain of integrin alpha5beta1-dependent cell adhesion on fibronectin: evidence for the negative regulation of fibronectin synergy site biological activity

Disintegrins are a family of potent inhibitors of cell-cell and cell-matrix adhesion. In this study we have identified a region of the disintegrin elegantin, termed the "linker domain" (amino acids 38-47), with inhibitory activity toward alpha(5)beta(1)-mediated cell adhesion on fibronectin (Fn). Using a chimeric structure-function approach in which sequences of the functionally distinct disintegrin kistrin were introduced into the elegantin template at targeted sites, a loss of inhibitory function toward alpha(5)beta(1)-mediated adhesion on Fn was observed when the elegantin linker domain was substituted. Subsequent analysis comparing the inhibitory efficacies of the panel of elegantin-kistrin chimeras toward CHO alpha(5) cell adhesion on recombinant Fn III(6-10) fragments showed that the loss of inhibitory activity associated with the disruption of the elegantin linker domain was dependent upon the presence of a functional Fn III(9) synergy site within the Fn III(6-10) substrate. This suggested that the elegantin linker domain inhibits primarily the activity of the Fn synergy domain in promoting alpha(5)beta(1) integrin-mediated cell adhesion. Construction of a cyclic peptide corresponding to the entire region of the elegantin linker domain showed that this domain has intrinsic alpha(5)beta(1) inhibitory activity comparable with the activity of the RGDS peptide. These data demonstrate a novel biological function for a disintegrin domain that antagonizes integrin-mediated cell adhesion.     

Intramolecular signaling in tandem-GAF domains from PDE5 and PDE10 studied with a cyanobacterial adenylyl cyclase reporter

The dimeric mammalian phosphodiesterases (PDEs) are regulated by N-terminal domains. In PDE5, the GAF-A subdomain of a GAF-tandem (GAF-A and -B) binds the activator cGMP and in PDE10 GAF-B binds cAMP. GAF-tandem chimeras of PDE5 and 10 in which the 36 aa linker helix between GAF-A and -B was swapped lost allosteric regulation of a reporter adenylyl cyclase. In 16 consecutive constructs we substituted the PDE10 linker with that from PDE5. An initial stretch of 10 amino acids coded for isoform specificity. A C240Y substitution uncoupled cyclase activity from regulation, whereas C240F, L or G did not. The C240Y substitution increased basal activity to stimulated levels. Notably, over the next 12 substitutions basal cyclase activity decreased linearly.Further targeted substitutions were based on homology modeling using the PDE2 structure. No combination of substitutions within the initial 10 linker residues caused loss of regulation. The full 10 aa stretch was required. Modeling indicated a potential interaction of the linker with a loop from GAF-A. To interrupt H-bonding a glycine substitution of the loop segment was generated. Despite reduction of basal activity, loss of regulation was maintained. Possibly, the orientation of the linker helix is determined by formation of the dimer at the initial linker segment. Downstream deflections of the linker helix may have caused loss of regulation.     

Probing conservation of HAMP linker structure and signal transduction mechanism through analysis of hybrid sensor kinases

The HAMP linker, a predicted structural element observed in many sensor kinases and methyl-accepting chemotaxis proteins, transmits signals between sensory input modules and output modules. HAMP linkers are located immediately inside the cytoplasmic membrane and are predicted to form two short amphipathic alpha-helices (AS-1 and AS-2) joined by an unstructured connector. HAMP linkers are found in the Escherichia coli nitrate- and nitrite-responsive sensor kinases NarX and NarQ (which respond to ligand by increasing kinase activity) and the sensor kinase CpxA (which responds to ligand by decreasing kinase activity). We constructed a series of hybrids with fusion points throughout the HAMP linker, in which the sensory modules of NarX or NarQ are fused to the transmitter modules of NarX, NarQ, or CpxA. A hybrid of the NarX sensor module and the CpxA HAMP linker and transmitter module (NarX-CpxA-1) responded to nitrate by decreasing kinase activity, whereas a hybrid in which the HAMP linker of NarX was replaced by that of CpxA (NarX-CpxA-NarX-1) responded to nitrate by increasing kinase activity. However, sequence variations between HAMP linkers do not allow free exchange of HAMP linkers or their components. Certain deletions in the NarX HAMP linker resulted in characteristic abnormal responses to ligand; similar deletions in the NarQ and NarX-CpxA-1 HAMP linkers resulted in responses to ligand generally similar to those seen in NarX. We conclude that the structure and action of the HAMP linker are conserved and that the HAMP linker transmits a signal to the output domain that ligand is bound.     

The Abl SH2-kinase linker naturally adopts a conformation competent for SH3 domain binding

The core of the Abelson tyrosine kinase (c-Abl) is structurally similar to Src-family kinases where SH3 and SH2 domains pack against the backside of the kinase domain in the down-regulated conformation. Both kinase families depend upon intramolecular association of SH3 with the linker joining the SH2 and kinase domains for suppression of kinase activity. Hydrogen deuterium exchange (HX) and mass spectrometry (MS) were used to probe intramolecular interaction of the c-Abl SH3 domain with the linker in recombinant constructs lacking the kinase domain. Under physiological conditions, the c-Abl SH3 domain undergoes partial unfolding, which is stabilized by ligand binding, providing a unique assay for SH3:linker interaction in solution. Using this approach, we observed dynamic association of the SH3 domain with the linker in the absence of the kinase domain. Truncation of the linker before W254 completely prevented cis-interaction with SH3, while constructs containing amino acids past this point showed SH3:linker interactions. The observation that the Abl linker sequence exhibits SH3-binding activity in the absence of the kinase domain is unique to Abl and was not observed with Src-family kinases. These results suggest that SH3:linker interactions may have a more prominent role in Abl regulation than in Src kinases, where the down-regulated conformation is further stabilized by a second intramolecular interaction between the C-terminal tail and the SH2 domain.     

The Extracytoplasmic Linker Peptide of the Sensor Protein SaeS Tunes the Kinase Activity Required for Staphylococcal Virulence in Response to Host Signals

Bacterial pathogens often employ two-component systems (TCSs), typically consisting of a sensor kinase and a response regulator, to control expression of a set of virulence genes in response to changing host environments. In Staphylococcus aureus, the SaeRS TCS is essential for in vivo survival of the bacterium. The intramembrane-sensing histidine kinase SaeS contains, along with a C-terminal kinase domain, a simple N-terminal domain composed of two transmembrane helices and a nine amino acid-long extracytoplasmic linker peptide. As a molecular switch, SaeS maintains low but significant basal kinase activity and increases its kinase activity in response to inducing signals such as human neutrophil peptide 1 (HNP1). Here we show that the linker peptide of SaeS controls SaeS’s basal kinase activity and that the amino acid sequence of the linker peptide is highly optimized for its function. Without the linker peptide, SaeS displays aberrantly elevated kinase activity even in the absence of the inducing signal, and does not respond to HNP1. Moreover, SaeS variants with alanine substitution of the linker peptide amino acids exhibit altered basal kinase activity and/or irresponsiveness to HNP1. Biochemical assays reveal that those SaeS variants have altered autokinase and phosphotransferase activities. Finally, animal experiments demonstrate that the linker peptide-mediated fine tuning of SaeS kinase activity is critical for survival of the pathogen. Our results indicate that the function of the linker peptide in SaeS is a highly evolved feature with very optimized amino acid sequences, and we propose that, in other SaeS-like intramembrane sensing histidine kinases, the extracytoplasmic linker peptides actively fine-control their kinases.     

The catalytic domain of a Piromyces rhizinflata cellulase expressed in Escherichia coli was stabilized by the linker peptide of the enzyme

Analysis of a carboxymethyl-cellulase clone isolated from the cDNA library of the ruminal fungus, Piromyces rhizinflata 2301, revealed that the clone encoded a polypeptide containing a cellulase catalytic domain, designated CelAcd. CelAcd was flanked by a 28-amino acid linker peptide at the N-terminus and linked to a dockerin domain by a 7-amino acid linker at the C-terminus. CelAcd showed homology with the family 5 of glycosyl hydrolases. CelAcd plus the linker peptides at both termini (designated CelcdN'C') was expressed in Escherichia coli and the purified enzyme was characterized. The feature of particular interest of CelcdN'C' was its bifunctional endo- and exo-glucanase activity, demonstrated by its ability to hydrolyse carboxymethyl cellulose (CMC) and pNP-beta-D-cellobioside. Furthermore, CelcdN'C' exhibited relatively high ability to degrade both microcrystalline Avicel and filter paper. CelcdN'C' also showed activity against barley beta-glucan, Lichenin and oat spelt xylan. The optimal activity conditions for CelcdN'C' with CMC as the substrate were pH 5.5 and 50 degrees C. Fifty percent of the enzyme activity was lost when CelcdN'C' was treated at 55 degrees C for 10 min. CelcdN'C' retained more than 10% enzyme activity after being heated under 90 degrees C for 10 min. Deletion of the N-terminal flanking linker of CelcdN'C' (the resulting protein was designated CelcdC') did not alter the enzymatic function of the catalytic domain. However, the thermal stability of CelcdC' was dramatically reduced. We conclude that the N-terminal flanking linker of CelAcd stabilizes the enzyme protein.     

Influence of linker unit on performance of palladium(II) coproporphyrin labelling reagent and its bioconjugates

In this paper we describe the preparation of a series of new phosphorescent labelling reagents, based on monosubstituted palladium(II) coproporphyrin‐I and the isothiocyanato reactive group. The labelling reagents differ with respect to the chemical composition of the linker unit that combines the reactive group and the porphyrin chromophore. Altogether, seven different labelling reagents are prepared. The new labelling reagents are conjugated with monoclonal mouse IgG to yield label conjugates with variable degrees of conjugation. The effect is studied of linker unit on: (a) the conjugation reaction kinetics; (b) the biological activity of the resulting IgG conjugates; and (c) the efficiency of phosphorescence emission. The results show that an increase in the length of the linker unit has a positive effect on both the reactivity of the label and the biological activity of the resulting conjugates. In addition, the results indicate that the labels with the most hydrophilic linker units exhibit the highest phosphorescence emission efficiencies. Copyright © 2003 John Wiley & Sons, Ltd.     

Mouse Dnmt3a preferentially methylates linker DNA and is inhibited by histone H1

In mammals, DNA methylation is crucial for embryonic development and germ cell differentiation. The DNA methylation patterns are created by de novo-type DNA methyltransferases (Dnmts) 3a and 3b. Dnmt3a is crucial for global methylation, including that of imprinted genes in germ cells. In eukaryotic nuclei, genomic DNA is packaged into multinucleosomes with linker histone H1, which binds to core nucleosomes, simultaneously making contacts in the linker DNA that separates adjacent nucleosomes. In the present study, we prepared oligonucleosomes from HeLa nuclei with or without linker histone H1 and used them as a substrate for Dnmt3a. Removal of histone H1 enhanced the DNA methylation activity. Furthermore, Dnmt3a preferentially methylated the linker between the two nucleosome core regions of reconstituted dinucleosomes, and the binding of histone H1 inhibited the DNA methylation activity of Dnmt3a towards the linker DNA. Since an identical amount of histone H1 did not inhibit the activity towards naked DNA, the inhibitory effect of histone H1 was not on the Dnmt3a catalytic activity but on its preferential location in the linker DNA of the dinucleosomes. The central globular domain and C-terminal tail of the histone H1 molecule were indispensable for inhibition of the DNA methylation activity of Dnmt3a. We propose that the binding and release of histone H1 from the linker portion of chromatin may regulate the local DNA methylation of the genome by Dnmt3a, which is expressed ubiquitously in somatic cells in vivo.     

The role of the linker between the SH2 domain and catalytic domain in the regulation and function of Src.

The crystal structures of the regulated Src and Hck tyrosine kinases show intramolecular interactions between the phosphorylated tail and the SH2 domain as well as between the SH3 domain, the SH2-catalytic domain linker (SH2-CD linker) and the catalytic domain. The relative contribution of these interactions to regulation of activity is poorly understood. Mutational analysis of Src and Lck revealed that interaction of the SH2-CD linker with the SH3 domain is crucial for regulation. Moreover, three sites of interaction of the linker with the catalytic domain, one at the beginning (Trp260) and two at the back of the small lobe, opposite the catalytic cleft (beta2/beta3 loop; alphaC/beta4 loop), impinge on Src activity. Other activating mutations map to the front of the catalytic domain in the loop preceding the alphaC-helix (beta3/alphaC loop). SH2-CD linker mutants are deregulated in mammalian cells but transform fibroblasts weakly, suggesting that the linker may bind cellular components. Interpretation of our results on the basis of the crystal structure of Src favours a model in which the correctly positioned SH2-CD linker exerts an inhibitory function on catalysis of Src family members by facilitating displacement of the alphaC-helix. This study may provide a template for the generation of deregulated versions of other protein kinases.     

Structure-activity relationship studies of S1P agonists with a dihydronaphthalene scaffold

Structure-activity relationship (SAR) of sphingosine-1-phosphate receptor agonists with a dihydronaphthalene scaffold was investigated. Compound 1 was modified to improve S1P(1) agonistic activity and in vivo peripheral lymphocyte lowering (PLL) activity without impairing selectivity over S1P(3) agonistic activity. A detailed SAR study of the terminal lipophilic part revealed that the introduction of substituents on the propylene linker and the terminal benzene ring influences in vitro and PLL activities. Compound 6n bearing a (S)-methyl group at the 2-position on the propylene linker and chlorine at the para-position on the terminal benzene ring showed potent hS1P(1) agonistic activity with excellent selectivity over hS1P(3) and in vivo PLL activity in mice.     

Reassembly of phospholipase C-beta2 from separated domains: analysis of basal and G protein-stimulated activities

Phosphatidylinositol-specific phospholipase C-betas (PLC-betas) are the only PLC isoforms that are regulated by G protein subunits. To further understand the regulation of PLC-beta(2) by G proteins and the functional roles of PLC-beta(2) structural domains, we tested whether the separately expressed amino and carboxyl halves of PLC-beta(2) could associate to form catalytically active enzymes as two polypeptides, and we explored how the complexes thus formed would be regulated by G protein betagamma subunits (Gbetagamma). We expressed cDNA constructs encoding PLC-beta(2) fragments of different lengths in COS-7 cells and demonstrated by coimmunoprecipitation that the coexpressed fragments could assemble and functionally reconstitute an active PLC-beta(2). The pleckstrin homology domain of PLC-beta(2) was required for its targeting to the membrane and for substrate hydrolysis. Reconstituted enzymes that contained the linker region that joins the two catalytic domains were as active or more active than the wild-type PLC-beta(2). When the linker region was removed, basal PLC-beta(2) enzymatic activity was increased further, suggesting that the linker region exerts an inhibitory effect on basal PLC-beta(2) activity. The reconstituted enzymes, like wild-type PLC-beta(2), were activated by Gbetagamma; when the C-terminal region was present in these constructs, they were also activated by Galpha(q). Gbetagamma and Galpha(q) activated these PLC-beta(2) constructs equally in the presence or absence of the linker region. We conclude that the linker region is an inhibitory element in PLC-beta(2) and that Gbetagamma and Galpha(q) do not stimulate PLC-beta(2) through easing the inhibition of enzymatic activity by the linker region.     

Functional analysis of the carbohydrate recognition domains and a linker peptide of galectin-9 as to eosinophil chemoattractant activity

Human galectin-9 is a beta-galactoside-binding protein consisting of two carbohydrate recognition domains (CRDs) and a linker peptide. We have shown that galectin-9 represents a novel class of eosinophil chemoattractants (ECAs) produced by activated T cells. A previous study demonstrated that the carbohydrate binding activity of galectin-9 is indispensable for eosinophil chemoattraction and that the N- and C-terminal CRDs exhibit comparable ECA activity, which is substantially lower than that of full-length galectin-9. In this study, we investigated the roles of the two CRDs in ECA activity in conjunction with the sugar-binding properties of the CRDs. In addition, to address the significance of the linker peptide structure, we compare the three isoforms of galectin-9, which only differ in the linker peptide region, in terms of ECA activity. Recombinant proteins consisting of two N-terminal CRDs (galectin-9NN), two C-terminal CRDs (galectin-9CC), and three isoforms of galectin-9 (galectin-9S, -9M, and -9L) were generated. All the recombinant proteins had hemagglutination activity comparable to that of the predominant wild-type galectin-9 (galectin-9M). Galectin-9NN and galectin-9CC induced eosinophil chemotaxis in a manner indistinguishable from the case of galectin-9M. Although the isoform of galectin-9 with the longest linker peptide, galectin-9L, exhibited limited solubility, the three isoforms showed comparable ECA activity over the concentration range tested. The interactions between N- and C-terminal CRDs and glycoprotein glycans and glycolipid glycans were examined using frontal affinity chromatography. Both CRDs exhibited high affinity for branched complex type sugar chain, especially for tri- and tetraantennary N-linked glycans with N-acetyllactosamine units, and the oligosaccharides inhibited the ECA activity at low concentrations. These results suggest that the N- and C-terminal CRDs of galectin-9 interact with the same or a closely related ligand on the eosinophil membrane when acting as an ECA and that ECA activity does not depend on a specific structure of the linker peptide.     

Modulation of a GEF switch: autoinhibition of the intrinsic guanine nucleotide exchange activity of p115-RhoGEF

p115-RhoGEF (p115) belongs to the family of RGS-containing guanine nucleotide exchange factors for Rho GTPases (RGS-RhoGEFs) that are activated by G12 class heterotrimeric G protein α subunits. All RGS-RhoGEFs possess tandemly linked Dbl-homology (DH) and plekstrin-homology (PH) domains, which bind and catalyze the exchange of GDP for GTP on RhoA. We have identified that the linker region connecting the N-terminal RGS-homology (RH) domain and the DH domain inhibits the intrinsic guanine nucleotide exchange (GEF) activity of p115, and determined the crystal structures of the DH/PH domains in the presence or absence of the inhibitory linker region. An N-terminal extension of the canonical DH domain (the GEF switch), which is critical to GEF activity, is well folded in the crystal structure of DH/PH alone, but becomes disordered in the presence of the linker region. The linker region is completely disordered in the crystal structure and partially disordered in the molecular envelope calculated from measurements of small angle x-ray scattering (SAXS). It is possible that Gα subunits activate p115 in part by relieving autoinhibition imposed by the linker region.     

The length of the linker between the epidermal growth factor‐like domains in factor VIIa is critical for a productive interaction with tissue factor

Formation of the factor VIIa (FVIIa)‐tissue factor (TF) complex triggers the blood coagulation cascade. Using a structure‐based rationale, we investigated how the length of the linker region between the two epidermal growth factor (EGF)‐like domains in FVIIa influences TF binding and the allosteric activity enhancement, as well as the interplay between the γ‐carboxyglutamic acid (Gla)‐containing and protease domains. Removal of two residues from the native linker was compatible with normal cofactor binding and accompanying stimulation of the enzymatic activity, as was extension by two (Gly‐Ser) residues. In sharp contrast, truncation by three or four residues abolished the TF‐mediated stabilization of the active conformation of FVIIa and abrogated TF‐induced activity enhancement. In addition, FVIIa variants with short linkers associated 80‐fold slower with soluble TF (sTF) as compared with wild‐type FVIIa, resulting in a corresponding increase in the equilibrium dissociation constant. Molecular modeling suggested that the shortest FVIIa variants would have to be forced into a tense and energetically unfavorable conformation in order to be able to interact productively with TF, explaining our experimental observations. We also found a correlation between linker length and the residual intrinsic enzymatic activity of Ca²⁺‐free FVIIa; stepwise truncation resulting in gradually higher activity with des(83–86)‐FVIIa reaching the level of Gla‐domainless FVIIa. The linker appears to determine the average distance between the negatively charged Gla domain and a structural element in the protease domain, presumably of opposite charge, and proximity has a negative impact on apo‐FVIIa activity.