Buried Cysteine Research Articles

PTM-Psi: A python package to facilitate the computational investigation of post-translational modification on protein structures and their impacts on dynamics and functions.

Post-translational modification (PTM) of a protein occurs after it has been synthesized from its genetic template, and involves chemical modifications of the protein's specific amino acid residues. Despite of the central role played by PTM in regulating molecular interactions, particularly those driven by reversible redox reactions, it remains challenging to interpret PTMs in terms of protein dynamics and function because there are numerous combinatorially enormous means for modifying amino acids in response to changes in the protein environment. In this study, we provide a workflow that allows users to interpret how perturbations caused by PTMs affect a protein's properties, dynamics, and interactions with its binding partners based on inferred or experimentally determined protein structure. This Python-based workflow, called PTM-Psi, integrates several established open-source software packages, thereby enabling the user to infer protein structure from sequence, develop force fields for non-standard amino acids using quantum mechanics, calculate free energy perturbations through molecular dynamics simulations, and score the bound complexes via docking algorithms. Using the S-nitrosylation of several cysteines on the GAP2 protein as an example, we demonstrated the utility of PTM-Psi for interpreting sequence-structure-function relationships derived from thiol redox proteomics data. We demonstrate that the S-nitrosylated cysteine that is exposed to the solvent indirectly affects the catalytic reaction of another buried cysteine over a distance in GAP2 protein through the movement of the two ligands. Our workflow tracks the PTMs on residues that are responsive to changes in the redox environment and lays the foundation for the automation of molecular and systems biology modeling.

Proteome-wide structural analysis identifies warhead- and coverage-specific biases in cysteine-focused chemoproteomics

Covalent drug discovery has undergone a resurgence over the past two decades and reactive cysteine profiling has emerged in parallel as a platform for ligand discovery through on- and off-target profiling; however, the scope of this approach has not been fully explored at the whole-proteome level. We combined AlphaFold2-predicted side-chain accessibilities for >95% of the human proteome with a meta-analysis of eighteen public cysteine profiling datasets, totaling 44,187 unique cysteine residues, revealing accessibility biases in sampled cysteines primarily dictated by warhead chemistry. Analysis of >3.5 million cysteine-fragment interactions further showed that hit elaboration and optimization drives increased bias against buried cysteine residues. Based on these data, we suggest that current profiling approaches cover a small proportion of potential ligandable cysteine residues and propose future directions for increasing coverage, focusing on high-priority residues and depth. All analysis and produced resources are freely available and extendable to other reactive amino acids.

Solution NMR studies reveal novel CLIC4 chaperone activity with respect to profilin-1.

Despite its name, chloride intracellular channel 4 (CLIC4) is a soluble enzyme that can catalyze glutathione-dependent thiol transfers. It is unclear how this enzymatic activity relates to its biological role in remodeling the actin cytoskeleton. Previous studies have shown that CLIC4 interacts with profilin-1, a critical regulator of actin polymerization. We confirmed a direct interaction, 170 ± 20 μM by microscale thermophoresis. However, using solution nuclear magnetic resonance (NMR) spectroscopy, we demonstrate that CLIC4 has a higher affinity for profilin when it is unfolded. Amide hydrogen exchange studies demonstrate that the N- and C-terminal helices of profilin readily unfold, whereas the central β-beta sheet core remains relatively intact. Interaction with CLIC4 increases amide exchange rates throughout profilin, indicating preferential stabilization of partially unfolded intermediates. We note that profilin is glutathionylated in proteomic studies of cells exposed to oxidative stress. Profilin is unfolded by glutathionylation of buried cysteine residues in its N- or C-terminal helices, and this disrupts its ability to bind actin, as measured by pyrene-actin polymerization assay. We are currently attempting to demonstrate that CLIC4 can reverse glutathionylation of profilin-1 and promote its refolding in cells and in vitro.

A conformational transition of the D′D3 domain primes von Willebrand factor for multimerization

AbstractVon Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the trans-Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the D′D3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose 2 buried cysteines, Cys1099 and Cys1142, that are vital for multimerization. By characterizing the conformational change at varying levels of force, we can quantify the kinetics of the transition and stability of the interface. We find a pronounced destabilization of the interface on lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the D′D3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII, providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the D′D3 domain in VWF biosynthesis and function, and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

Improvement of Selenomonas ruminantium β-xylosidase thermal stability by replacing buried free cysteines via site directed mutagenesis

β-xylosidase is an essential enzyme for breakdown of xylan to d-xylose. It has a significant potential application value for medicine, food, paper and pulp, and biofuel industries. Due to the negative consequences caused by buried free cysteine residues, mutational substitution of such residues is often accompanied by a notable increase in thermal stability. To characterize the role of cysteine residues in the structure, function and stability of Selenomonas ruminantium β-d-Xylosidase (SXA), we prepared and evaluated wild-type and four cysteines- deficient SXA proteins. Buried cysteine residues were replaced with. In comparison with the wild-type, the Km values of the mutants remained relatively constant while their kcat values decreased. The C101V and C286V displayed higher thermal stability than the wild-type at 55 and 60 °C. Conformational changes of the secondary and tertiary structure as derived from circular dichroism and fluorescence spectroscopy revealed that changing a buried cysteine to a hydrophobic residue could lead to an increase in thermal stability with minimal perturbation of the wild-type protein structure. In addition to experimental methods, the stability of WT SXA and C101V and C286V mutants at 333 K was also studied by MD simulation. Our theoretical data had a good agreement with the experimental results.

Interplay of cysteine exposure and global protein dynamics in small-molecule recognition by a regulator of G-protein signaling protein.

Regulator of G protein signaling (RGS) proteins play a pivotal role in regulation of G protein-coupled receptor (GPCR) signaling and are therefore becoming an increasingly important therapeutic target. Recently discovered thiadiazolidinone (TDZD) compounds that target cysteine residues have shown different levels of specificities and potencies for the RGS4 protein, thereby suggesting intrinsic differences in dynamics of this protein upon binding of these compounds. In this work, we investigated using atomistic molecular dynamics (MD) simulations the effect of binding of several small-molecule inhibitors on perturbations and dynamical motions in RGS4. Specifically, we studied two conformational models of RGS4 in which a buried cysteine residue is solvent-exposed due to side-chain motions or due to flexibility in neighboring helices. We found that TDZD compounds with aromatic functional groups perturb the RGS4 structure more than compounds with aliphatic functional groups. Moreover, small-molecules with aromatic functional groups but lacking sulfur atoms only transiently reside within the protein and spontaneously dissociate to the solvent. We further measured inhibitory effects of TDZD compounds using a protein-protein interaction assay on a single-cysteine RGS4 protein showing trends in potencies of compounds consistent with our simulation studies. Thermodynamic analyses of RGS4 conformations in the apo-state and on binding to TDZD compounds revealed links between both conformational models of RGS4. The exposure of cysteine side-chains appears to facilitate initial binding of TDZD compounds followed by migration of the compound into a bundle of four helices, thereby causing allosteric perturbations in the RGS/Gα protein-protein interface.

Role of Protein Dynamics in Selectivity of Thiadiazolidinone Inhibition of RGS Proteins

BackgroundRegulator of G‐protein Signaling (RGS) proteins play a critical role in GPCR (G‐protein Coupled Receptor) signaling by binding to active, GTP‐bound Gα subunits and accelerating GTP hydrolysis, thus terminating signaling. There are multiple RGS isoforms with signature tissue distributions, so by targeting RGS proteins in an isoform‐specific manner, better tissue specificity of pharmacological effects may be achieved than by global application of a GPCR agonist. Thiadiazolidinones (TDZDs) are a series of compounds that inhibit RGS proteins by covalent modification of cysteine residues. They are very potent against RGS4, but also act less potently on several other cysteine‐containing RGS proteins. RGS4, RGS8, and RGS19, three proteins that are inhibited by TDZDs, share a cysteine on the α4 helix of the RGS homology domain. RGS19 has only this cysteine, while RGS4 and RGS8 have additional cysteines, including a shared one on the α6‐α7 interhelical loop. Interestingly, these shared cysteines are not exposed to solvent in crystal structures.HypothesisDifferences in potency due to covalent modification of the shared cysteine at the α4 helix of RGS proteins is driven by differences in flexibility.ApproachThe rate of covalent modification at individual cysteines can be determined by treating proteins with iodoacetamide, digestion, and detection of adduct by mass spectrometry. In addition, the effect of modification at different cysteines on protein function can be evaluated by measuring TDZD inhibition of binding between Gα and cysteine mutants of RGS proteins.ResultsDeuterium exchange and molecular dynamics studies have shown differential flexibility in key regions of these RGS proteins, which may be necessary for TDZD access to buried cysteines. RGS4 is more potently inhibited by CCG‐50014 (IC50: 81 nM) than RSG19 (IC50: 1.1 μM) or RGS8 (IC50: 1.8 μM). However, when RGS4 or RGS8 are mutated to contain only the α4 helical cysteine, the potency of TDZD inhibition is drastically reduced to below that of RGS19 (RGS4 IC50: 8.5 μM, RGS8 IC50 >100 μM). In accordance with previous molecular simulation data showing this α4 cysteine to be least solvent accessible in RGS4, it had a lower degree of covalent adduct formation by iodoacetamide than other cysteines.ConclusionThese results indicate that while additional cysteines in RGS4 may be responsible for its high potency of inhibition by TDZDs, differences in the rate of covalent modification and subsequent potency of inhibition at the conserved α4 cysteine is driven by differences in RGS protein flexibility.Support or Funding InformationNIH T32 GM092715NSF 1507588This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reagent enables new protein modification

Chemists modify protein sidechains to modulate the biomolecules’ functions. Researchers are usually stuck modifying cysteines because the amino acid’s sulfide side chain makes it the most reactive amino acid. But cysteines are relatively rare, representing ~1.9% of the amino acids in human proteins. Cysteines can be installed using genetic engineering, but researchers would like to modify fully functional native proteins. An international team of researchers led by Goncalo J. L. Bernardes of the University of Cambridge and the Institute of Molecular Medicine Lisbon and Gonzalo Jimenez-Oses of the University of Rioja has now discovered a sulfonyl acrylate reagent that should give researchers more options for modifying proteins. The reagent selectively targets only the most reactive lysine in a protein—not other lysines or buried cysteines (J. Am. Chem. Soc. 2018, DOI: 10.1021/jacs.7b12874). Lysines are nucleophilic and much more abundant than cysteines, representing ~5.9% of the amino acids in human protei...

Differential Protein Dynamics of Regulators of G-Protein Signaling: Role in Specificity of Small-Molecule Inhibitors

Small-molecule inhibitor selectivity may be influenced by variation in dynamics among members of a protein family. Regulator of G-protein Signaling (RGS) proteins are a family that plays a key role in G-Protein Coupled Receptor (GPCR) signaling by binding to active Gα subunits and accelerating GTP hydrolysis, thereby terminating activity. Thiadiazolidinones (TDZDs) inhibit the RGS-Gα interaction by covalent modification of cysteine residues in RGS proteins. Some differences in specificity may be explained by differences in the complement of cysteines among RGS proteins. However, key cysteines shared by RGS proteins inhibited by TDZDs are not exposed on the protein surface, and differences in potency exist among RGS proteins containing only buried cysteines. We hypothesize that differential exposure of buried cysteine residues among RGS proteins partially drives TDZD selectivity. Hydrogen-deuterium exchange (HDX) studies and molecular dynamics (MD) simulations were used to probe the dynamics of RGS4, RGS8, and RGS19, three RGS proteins inhibited at a range of potencies by TDZDs. When these proteins were mutated to contain a single, shared cysteine, RGS19 was found to be most potently inhibited. HDX studies revealed differences in α4 and α6 helix flexibility among RGS isoforms, with particularly high flexibility in RGS19. This could cause differences in cysteine exposure and lead to differences in potency of TDZD inhibition. MD simulations of RGS proteins revealed motions that correspond to solvent exposure observed in HDX, providing further evidence for a role of protein dynamics in TDZD selectivity.

An oxygen-sensitive toxin-antitoxin system.

The Hha and TomB proteins from Escherichia coli form an oxygen-dependent toxin–antitoxin (TA) system. Here we show that YmoB, the Yersinia orthologue of TomB, and its single cysteine variant [C117S]YmoB can replace TomB as antitoxins in E. coli. In contrast to other TA systems, [C117S]YmoB transiently interacts with Hha (rather than forming a stable complex) and enhances the spontaneous oxidation of the Hha conserved cysteine residue to a -SOxH-containing species (sulfenic, sulfinic or sulfonic acid), which destabilizes the toxin. The nuclear magnetic resonance structure of [C117S]YmoB and the homology model of TomB show that the two proteins form a four-helix bundle with a conserved buried cysteine connected to the exterior by a channel with a diameter comparable to that of an oxygen molecule. The Hha interaction site is located on the opposite side of the helix bundle.

Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for “Second Generation” Therapeutic Application

Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys-free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. Here, we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased and is equivalent to wild-type FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys-free FGF-1 mutant as an advantageous “second generation” form of FGF-1 for therapeutic application.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Unique Conformational and Chemical Transformations Occurring upon [4Fe-4S] Cluster Binding in the Type 2 L-Serine Dehydratase from Legionella pneumophila.

The type 2 L-serine dehydratase from Legionella pneumophila (lpLSD) contains a [4Fe-4S](2+) cluster that acts as a Lewis acid to extract the hydroxyl group of L-serine during the dehydration reaction. Surprisingly, the crystal structure shows that all four of the iron atoms in the cluster are coordinated with protein cysteinyl residues and that the cluster is buried and not exposed to solvent. If the crystal structure of lpLSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter. Furthermore, repair of the oxidized protein when the cluster has degraded would presumably entail exposure of the buried cysteine ligands. Thus, the conformation required for the substrate to enter may be similar to those required for a new cluster to enter the active site. To address this, hydrogen-deuterium exchange combined with mass spectrometry (HDX MS) was used to probe the conformational changes that occur upon oxidative degradation of the Fe-S cluster. The regions that show the most significant differential HDX are adjacent to the cluster location in the holoenzyme or connect regions that are adjacent to the cluster. The observed decrease in flexibility upon cluster binding provides direct evidence that the "tail-in-mouth" conformation observed in the crystal structure also occurs in solution and that the C-terminal peptide is coordinated to the [4Fe-4S] cluster in a precatalytic conformation. This observation is consistent with the requirement of an activation step prior to catalysis and the unusually high level of resistance to oxygen-induced cluster degradation. Furthermore, peptide mapping of the apo form under nonreducing conditions revealed the formation of disulfide bonds between C396 and C485 and possibly between C343 and C385. These observations provide a picture of how the cluster loci are stabilized and poised to receive the cluster in the apo form and the requirement for a reduction step during cluster formation.

Regulators of G‐protein Signaling Proteins: Conformational Dynamics and Allosteric Control

Regulators of G-protein signaling (RGS) proteins are intracellular proteins that negatively modulate the signaling pathways of G-protein coupled receptors (GPCRs). Because RGS proteins directly bind to activated G-proteins and accelerate the rate of GTP-hydrolysis, they have emerged as potential drug targets for regulating GPCR signaling. While conventional approaches have been focused on directly targeting RGS-Gα protein-protein interaction using small molecules, new approaches that target allosteric sites are emerging. In this work, we have studied using atomistic simulations the mechanism of action of a small molecule that binds, with differing affinity, to specific cysteine residues on two structurally homologous RGS proteins, RGS4 and RGS8. We find that each RGS protein has flexibility in a pair of helices that allows small molecule to access buried cysteine residues. Such flexibility only became apparent on application of enhanced sampling simulations on apo-RGS structures. Moreover, we observe that the small molecule can spontaneously bind to “conformationally-open” RGS proteins, but only associates with different surface sites in “conformationally-closed” RGS proteins. The binding of small molecule to a buried cysteine residue on each protein also appears to perturb the protein-protein interface in each RGS-Gα complex via an allosteric mechanism.

Oxidative Stress Regulates Titin Elasticity by Affecting Ig-Domain Stability

Background: The elasticity of titin is regulated through several mechanisms, including isoform switching and phosphorylation of unique spring elements (N2-Bus, PEVK). However, the titin springs consist mainly of immunoglobulin-like (Ig) domains, which are centrally involved in the molecular mechanism of titin elasticity. Passive force-regulating mechanisms targeting the Ig-domains of titin have not been described.Methods and Results: We have elucidated a novel oxidative stress-related mechanism regulating muscle elasticity by altering the stability of titin-Ig domains (Alegre-Cebollada et al., Cell. 2014;156:1235-46). Using single-molecule AFM force spectroscopy, force measurements of isolated skinned human myocytes, and redox proteomics, we show that I-band Ig-domains of titin are weakened by oxidative modification of cryptic cysteines. We demonstrate that mechanical unfolding of these Ig domains exposes hidden cysteines, which now become accessible to disulfide bonding or S-glutathionylation in the presence of millimolar concentrations of oxidized glutathione (GSSG). In the AFM experiments, the cysteines of unfolded titin-Ig domains preferentially formed mixed disulfides with glutathione, which prevented the refolding of these domains. Oxidation by GSSG substantially reduced the passive tension of stretched human myocytes, and the effect was fully reversible with the incubation of reduced glutathione. Exposing perfused mouse hearts to oxidative stress (0.1 mM H2O2) revealed that Ig-domains from I-band titin are preferential targets of oxidation, as monitored using ICAT labeling/mass spectrometry.Conclusions: Titin elasticity in striated muscle is modulated by oxidative stress through reversible weakening of Ig-domain stability via S-glutathionylation of buried cysteines. These titin Ig domains could also represent individual mechanosensors, whose mechanical properties determine mechano-chemical signaling processes in stressed myocytes.

Computational Design of Repeat-Proteins with a Predefined Geometry

New protein design methods are needed to further improve the development of protein-binding scaffolds. Repeat proteins are linear tandem arrays of structurally similar building blocks, and they are established platforms for engineering proteins inhibitors and biosensors. However, current sequence-based engineering approaches lack the possibility of customizing the overall shape of a binder to its target molecule. Structure-based protein design offers a possibility of optimizing the overall shape of engineered binding scaffolds to better match their targets. We developed a protocol for the computational design of shape-optimized binding scaffolds that can better match their targets. By combining sequence optimization of existing repeats and de novo design of capping structures, we designed leucine-rich repeat (LRR) proteins where the building blocks assemble with a novel geometry. We validated the geometric design approach by engineering an artificial donut-like ring structure constructed from ten self-compatible repeats. Characterization of the design constructs revealed that buried cysteines play a central role for stability and folding cooperativity in certain LRR proteins. This may be used to selectively stabilize or destabilize specific parts of a protein. The computational procedure may now be employed to develop repeat proteins with various geometrical shapes for applications where greater control of the interface geometry is desired.

S-Glutathionylation of Cryptic Cysteines Enhances Titin Elasticity by Blocking Protein Folding

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric-oxide-signaling cascade that increases cardiac muscle elasticity. However, it is unknown how S-glutathionylation may regulate the elasticity of titin and cardiac tissue. Here, we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore, we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechanochemical modulation of the elasticity of human cardiomyocytes. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.

Large-Scale Modulation of Titin Elasticity by S-Glutathionylation of Cryptic Cysteines

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole, and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric oxide signaling cascade that increases cardiac muscle elasticity. However, it is unknown whether S-glutathionylation regulates the elasticity of titin and cardiac tissue. Here, we use homology modeling techniques to show that most immunoglobulin (Ig) domains in the elastic I-band of titin contain cryptic cysteines, which are potential targets of S-glutathionylation triggered by physiological mechanical protein unfolding in the heart. We choose I91 as a representative Ig domain of titin to investigate the effects of S-glutathionylation in the elasticity of the protein. Using single-molecule force-clamp spectroscopy, we demonstrate that mechanical unfolding of I91 exposes two buried cysteine residues, which then can be S-glutathionylated by oxidized glutathione in the solution. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of I91, which unfolds at a rate two orders of magnitude faster following S-glutathionylation. In addition, S-glutathionylation severely compromises the ability of I91 to fold. Both effects, which are fully reversed by the enzyme glutaredoxin, soften the I91 domain. When extrapolated to all the Ig domains in the I-band, our observations predict that S-glutathionylation can trigger a highly extensible state of titin. Indeed, we show that S-glutathionylation of cryptic cysteines in titin mediates mechano-chemical modulation of the elasticity of human cardiomyocytes. Monte Carlo simulations illustrate that large-scale regulation of the elasticity of titin through posttranslational modification of cryptic cysteines can be achieved on time scales of minutes to hours. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.

Tuning of Thioredoxin Redox Properties by Intramolecular Hydrogen Bonds

Thioredoxin-like proteins contain a characteristic C-x-x-C active site motif and are involved in a large number of biological processes ranging from electron transfer, cellular redox level maintenance, and regulation of cellular processes. The mechanism for deprotonation of the buried C-terminal active site cysteine in thioredoxin, necessary for dissociation of the mixed-disulfide intermediate that occurs under thiol/disulfide mediated electron transfer, is not well understood for all thioredoxin superfamily members. Here we have characterized a 8.7 kD thioredoxin (BC3987) from Bacillus cereus that unlike the typical thioredoxin appears to use the conserved Thr8 side chain near the unusual C-P-P-C active site to increase enzymatic activity by forming a hydrogen bond to the buried cysteine. Our hypothesis is based on biochemical assays and thiolate pKa titrations where the wild type and T8A mutant are compared, phylogenetic analysis of related thioredoxins, and QM/MM calculations with the BC3987 crystal structure as a precursor for modeling of reduced active sites. We suggest that our model applies to other thioredoxin subclasses with similar active site arrangements.

Identification of Serpin Determinants of Specificity and Selectivity for Furin Inhibition through Studies of α1PDX (α1-Protease Inhibitor Portland)-Serpin B8 and Furin Active-site Loop Chimeras

α1-Protease inhibitor Portland (α1PDX) is an engineered serpin family inhibitor of the proprotein convertase (PC), furin, that exhibits high specificity but limited selectivity for inhibiting furin over other PC family proteases. Here, we characterize serpin B8, a natural inhibitor of furin, together with α1PDX-serpin B8 and furin-PC chimeras to identify determinants of serpin specificity and selectivity for furin inhibition. Replacing reactive center loop (RCL) sequences of α1PDX with those of serpin B8 demonstrated that both the P4-P1 RXXR recognition sequence as well as the P1'-P5' sequence are critical determinants of serpin specificity for furin. Alignments of PC catalytic domains revealed four variable active-site loops whose role in furin reactivity with serpin B8 was tested by engineering furin-PC loop chimeras. The furin(298-300) loop but not the other loops differentially affected furin reactivity with serpin B8 and α1PDX in a manner that depended on the serpin RCL-primed sequence. Modeling of the serpin B8-furin Michaelis complex identified serpin exosites in strand 3C close to the 298-300 loop whose substitution in α1PDX differentially affected furin reactivity depending on the furin loop and serpin RCL-primed sequences. These studies demonstrate that RCL-primed residues, strand 3C exosites, and the furin(298-300) loop are critical determinants of serpin reactivity with furin, which may be exploited in the design of specific and selective α1PDX inhibitors of PCs.

Probing the Folded State of Fibronectin Type III Domains in Stretched Fibrils by Measuring Buried Cysteine Accessibility

Fibronectin (FN) is an extracellular matrix protein that is assembled into fibrils by cells during tissue morphogenesis and wound healing. FN matrix fibrils are highly elastic, but the mechanism of elasticity has been debated: it may be achieved by mechanical unfolding of FN-III domains or by a conformational change of the molecule without domain unfolding. Here, we investigate the folded state of FN-III domains in FN fibrils by measuring the accessibility of buried cysteines. Four of the 15 FN-III domains (III-2, -3, -9, and -11) appear to unfold in both stretched fibrils and in solution, suggesting that these domains spontaneously open and close even in the absence of tension. Two FN-III domains (III-6 and -12) appear to unfold only in fibrils and not in solution. These results suggest that domain unfolding can at best contribute partially to the 4-fold extensibility of fibronectin fibrils.