Understanding the specificity of serpin–protease complexes through interface analysis

Serpins such as antithrombin, heparin cofactor II, plasminogen activator inhibitor, antitrypsin, antichymotrypsin, and neuroserpin are involved in important biological processes by inhibiting specific serine proteases. Initially, the protease recognizes the mobile reactive loop of the serpin eliciting conformational changes, where the cleaved loop together with the protease inserts into β-sheet A, translocating the protease to the opposite side of inhibitor leading to its inactivation. Serpin interaction with proteases is governed mainly by the reactive center loop residues (RCL). However, in some inhibitory serpins, exosite residues apart from RCL have been shown to confer protease specificity. Further, this forms the basis of multi-specificity of some serpins, but the residues and their dimension at interface in serpin-protease complexes remain elusive. Here, we present a comprehensive structural analysis of the serpin-protease interfaces using bio COmplexes COntact MAPS (COCOMAPS), PRotein Interface Conservation and Energetics (PRICE), and ProFace programs. We have carried out interface, burial, and evolutionary analysis of different serpin-protease complexes. Among the studied complexes, non-inhibitory serpins exhibit larger interface region with greater number of residue involvement as compared to the inhibitory serpins. On comparing the multi-specific serpins (antithrombin and antitrypsin), a difference in the interface area and residue number was observed, suggestive of a differential mechanism of action of these serpins in regulating their different target proteases. Further, detailed study of these multi-specific serpins listed few essential residues (common in all the complexes) and certain specificity (unique to each complex) determining residues at their interfaces. Structural mapping of interface residues suggested that individual patches with evolutionary conserved residues in specific serpins determine their specificity towards a particular protease.     

Interaction of Duodenase with Serpins from Human Blood Serum

The interaction of duodenase, a new serine protease from a small group of Janus-faced proteases, with serpins, ₁-protease inhibitor (₁-PI) and antichymotrypsin (ACT) from human blood serum, was studied. The stoichiometry of the inhibition process was found to be 1.2 and 1.3 mol/mol for ₁-PI and ACT, respectively. The presence of a stable enzyme–inhibitory complex duodenase–₁-PI was confirmed by SDS-PAGE. The formation of the duodenase–ACT complex was not demonstrated; instead, the band of the cleaved inhibitor indicated the ACT hydrolysis. The suicide mechanism of the duodenase interaction with the human blood serpins was proved. The association rate constants (k × 10⁵, ^–1 s^–1) were 2.4 ± 0.3 × 10⁵ for ₁-PI and 3.0 ± 0.4 × 10⁵ for ACT. These results indicate the possibility of the regulation of duodenase activity by endogenous serpins.     

Engineering trypsin for inhibitor resistance

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [k_cat/K_M = (1.2 ± 0.3) × 10⁷ M⁻¹ s⁻¹. Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.     

Metals affect the structure and activity of human plasminogen activator inhibitor-1. II. Binding affinity and conformational changes

Human plasminogen activator inhibitor type 1 (PAI-1) is a serine protease inhibitor with a metastable active conformation. The lifespan of the active form of PAI-1 is modulated via interaction with the plasma protein, vitronectin, and various metal ions. These metal ions fall into two categories: Type I metals, including calcium, magnesium, and manganese, stabilize PAI-1 in the absence of vitronectin, whereas Type II metals, including cobalt, copper, and nickel, destabilize PAI-1 in the absence of vitronectin, but stabilize PAI-1 in its presence. To provide a mechanistic basis for understanding the unusual modulation of PAI-1 structure and activity, the binding characteristics and conformational effects of these two types of metals were further evaluated. Steady-state binding measurements using surface plasmon resonance indicated that both active and latent PAI-1 exhibit a dissociation constant in the low micromolar range for binding to immobilized nickel. Stopped-flow measurements of approach-to-equilibrium changes in intrinsic protein fluorescence indicated that the Type I and Type II metals bind in different modes that induce distinct conformational effects on PAI-1. Changes in the observed rate constants with varying concentrations of metal allowed accurate determination of binding affinities for cobalt, nickel, and copper, yielding dissociation constants of ～40, 30, and 0.09 μM, respectively. Competition experiments that tested effects on PAI-1 stability were consistent with these measurements of affinity and indicate that copper binds tightly to PAI-1.     

How small peptides block and reverse serpin polymerisation

Many of the late-onset dementias, including Alzheimer's disease and the prion encephalopathies, arise from the aberrant aggregation of individual proteins. The serpin family of serine protease inhibitors provides a well-defined structural example of such pathological aggregation, as its mutant variants readily form long-chain polymers, resulting in diseases ranging from thrombosis to dementia. The intermolecular linkages result from the insertion of the reactive site loop of one serpin molecule into the middle strand (s4A) position of the A beta-sheet of another molecule. We define here the structural requirements for small peptides to competitively bind to and block the s4A position to prevent this intermolecular linkage and polymerisation. The entry and anchoring of blocking-peptides is facilitated by the presence of a threonine which inserts into the site equivalent to P8 of s4A. But the critical requirement for small blocking-peptides is demonstrated in crystallographic structures of the complexes formed with selected tri- and tetrapeptides. These structures indicate that the binding is primarily due to the insertion of peptide hydrophobic side-chains into the P4 and P6 sites of s4A. The findings allow the rational design of synthetic blocking-peptides small enough to be suitable for mimetic design. This is demonstrated here with a tetrapeptide that preferentially blocks the polymerisation of a pathologically unstable serpin commonly present in people of European descent.     

Mutagenesis of the bovSERPINA3-3 demonstrates the requirement of aspartate-371 for intermolecular interaction and formation of dimers

The family of serpins is known to fold into a metastable state that is required for the proteinase inhibition mechanism. One of the consequences of this conformational flexibility is the tendency of some mutated serpins to form polymers, which occur through the insertion of the reactive center loop of one serpin molecule into the A-sheet of another. This "A-sheet polymerization" has remained an attractive explanation for the molecular mechanism of serpinopathies. Polymerization of serpins can also take place in vitro under certain conditions (e.g., pH or temperature). Surprisingly, on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, bovSERPINA3-3 extracted from skeletal muscle or expressed in Escherichia coli was mainly observed as a homodimer. Here, in this report, by site-directed mutagenesis of recombinant bovSERPINA3-3, with substitution D371A, we demonstrate the importance of D371 for the intermolecular linkage observed in denaturing and reducing conditions. This residue influences the electrophoretic and conformational properties of bovSERPINA3-3. By structural modeling of mature bovSERPINA3-3, we propose a new "non-A-sheet swap" model of serpin homodimer in which D371 is involved at the molecular interface.     

Effects of antimicrobial peptides of neutrophils on tumor and normal host cells in culture

We carried out a study of the effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine Bac5 on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic β-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic effects. Bac5 belongs to a group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a nonlytic mechanism. We have shown that while PG1 exerted distinct and fast cytotoxic effects towards most of used tumor cells being in a lesser degree toxic for nontransformed host cells; the proline-rich peptide Bac5 possessed modest cytotoxic activity for all tested cells. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as was previously established for human defensins by Panyutich at al., 1995). Pre-incubation of PG1 with α1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of α1-antitrypsin was reduced after the interaction of the serpin with PG1 (while Bac5 did not affect the antiprotease activity of α1-antitrypsin). We used BODIPY FL-tagged PG1 and Bac5 to study the internalization of the labeled peptides into target cells and their intracellular distribution by confocal microscopy. Bac5-BODIPY (at 5 μ M) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations (1—3 μM) was also able to enter the cells without their damaging. By using flow cytometry we showed that lowering a temperature to 4°C caused a significant decrease in the uptake into K562 and U937 cells for both Bac5-BODIPY and PG1-BODIPY. A decline of target cells metabolism also diminished the process of both peptides internalization but for a lesser degree. In the presence of endocytosis inhibitors the penetration of Bac5-BODIPY and PG1-BODIPY into K562 cells was also reduced, but not completely abolished, suggesting that along with endocytosis process some direct penetration of the peptides across cell membranes takes place. The ability of the peptides to internalize into eukaryotic cells may contribute to the idea of participation of AMPs in varied intracellular events, occurring in normal or malignant host cells, for instance, in the modulation of intracellular serpins activity.     

Detection of serpins involved in cellular immune response of Rhipicephalus microplus challenged with fungi

The understanding of tick physiology and immune system is important to improve the effective control of this ectoparasite. Invertebrates' innate immune response is activated when the organism is challenged with pathogens. The present study describes the changes of serine proteinase inhibitors (serpins) and in the number of circulating haemocytes involved in cellular immune defence of Rhipicephalus microplus engorged females challenged with the entomopathogenic fungi Metarhizium anisopliae or Beauveria bassiana, or with the non-entomopathogenic fungus Fusarium oxysporum. The cell-free haemolymph was separated from haemocytes by centrifugation and cells were re-suspended in phosphate buffer pH 7.2. The proteins of haemocytes were analysed by SDS-PAGE and the segments of the 1D gel were submitted to protein digestion with trypsin. The peptides were analysed by liquid chromatography coupled with electrospray tandem mass spectrometry (LC-ESI-MS/MS). The analysis by mass spectrometry allowed the identification of several proteins through the search in the database built based on public banks of Ixodidae and Argasidae. In haemocytes, many proteins were identified highlighting serpins. The results showed that the entomopathogenic fungi M. anisopliae or B. bassiana reduced the amount of serpins, while F. oxysporum increased. The present study reports, for the first time, the variation of serpins in haemocytes of R. microplus engorged females infected by fungi.