Arg236 in human chymotrypsin B2 (CTRB2) is a key determinant of high enzyme activity, trypsinogen degradation capacity, and protection against pancreatitis.

Pancreatic chymotrypsins (CTRs) are digestive proteases that in humans include CTRB1, CTRB2, CTRC, and CTRL. The highly similar CTRB1 and CTRB2 are the products of gene duplication. A common inversion at the CTRB1-CTRB2 locus reverses the expression ratio of these isoforms in favor of CTRB2. Carriers of the inversion allele are protected against the inflammatory disorder pancreatitis presumably via their increased capacity for CTRB2-mediated degradation of harmful trypsinogen. To reveal the protective molecular determinants of CTRB2, we compared enzymatic properties of CTRB1, CTRB2, and bovine CTRA (bCTRA). By evolving substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) inhibitory loop variants against the chymotrypsins, we found that the substrate binding groove of the three enzymes had overlapping specificities. Based on the selected sequences, we produced eight SGPI-2 variants. Remarkably, CTRB2 and bCTRA bound these inhibitors with significantly higher affinity than CTRB1. Moreover, digestion of peptide substrates, beta casein, and human anionic trypsinogen unequivocally confirmed that CTRB2 is a generally better enzyme than CTRB1 while the potency of bCTRA lies between those of the human isoforms. Unexpectedly, mutation D236R alone converted CTRB1 to a CTRB2-like high activity protease. Modeling indicated that in CTRB1 Met210 partially obstructed the substrate binding groove, which was relieved by the D236R mutation. Taken together, we identify CTRB2 Arg236 as a key positive determinant, while CTRB1 Asp236 as a negative determinant for chymotrypsin activity. These findings strongly support the concept that in carriers of the CTRB1-CTRB2 inversion allele, the superior trypsinogen degradation capacity of CTRB2 protects against pancreatitis.

Directed Evolution-Driven Increase of Structural Plasticity Is a Prerequisite for Binding the Complement Lectin Pathway Blocking MASP-Inhibitor Peptides.

MASP-1 and MASP-2 are key activator proteases of the complement lectin pathway. The first specific mannose-binding lectin-associated serine protease (MASP) inhibitors had been developed from the 14-amino-acid sunflower trypsin inhibitor (SFTI) peptide by phage display, yielding SFTI-based MASP inhibitors, SFMIs. Here, we present the crystal structure of the MASP-1/SFMI1 complex that we analyzed in comparison to other existing MASP-1/2 structures. Rigidified backbone structure has long been accepted as a structural prerequisite for peptide inhibitors of proteases. We found that a hydrophobic cluster organized around the P2 Thr residue is essential for the structural stability of wild-type SFTI. We also found that the same P2 Thr prevents binding of the rigid SFTI-like peptides to the substrate-binding cleft of both MASPs as the cleft is partially blocked by large gatekeeper enzyme loops. Directed evolution removed this obstacle by replacing the P2 Thr with a Ser, providing the SFMIs with high-degree structural plasticity, which proved to be essential for MASP inhibition. To gain more insight into the structural criteria for SFMI-based MASP-2 inhibition, we systematically modified MASP-2-specific SFMI2 by capping its two termini and by replacing its disulfide bridge with varying length thioether linkers. By doing so, we also aimed to generate a versatile scaffold that is resistant to reducing environment and has increased stability in exopeptidase-containing biological environments. We found that the reduction-resistant disulfide-substituted l-2,3-diaminopropionic acid (Dap) variant possessed near-native potency. As MASP-2 is involved in the life-threatening thrombosis in COVID-19 patients, our synthetic, selective MASP-2 inhibitors could be relevant coronavirus drug candidates.

Altered glycosylation of IgG4 promotes lectin complement pathway activation in anti-PLA2R1-associated membranous nephropathy.

Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1-positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

Novel MASP-2 inhibitors developed via directed evolution of human TFPI1 are potent lectin pathway inhibitors.

The lectin pathway (LP) of the complement system is an important antimicrobial defense mechanism, but it also contributes significantly to ischemia reperfusion injury (IRI) associated with myocardial infarct, stroke, and several other clinical conditions. Mannan-binding lectin-associated serine proteinase 2 (MASP-2) is essential for LP activation, and therefore, it is a potential drug target. We have previously developed the first two generations of MASP-2 inhibitors by in vitro evolution of two unrelated canonical serine proteinase inhibitors. These inhibitors were selective LP inhibitors, but their nonhuman origin rendered them suboptimal lead molecules for drug development. Here, we present our third-generation MASP-2 inhibitors that were developed based on a human inhibitor scaffold. We subjected the second Kunitz domain of human tissue factor pathway inhibitor 1 (TFPI1 D2) to directed evolution using phage display to yield inhibitors against human and rat MASP-2. These novel TFPI1-based MASP-2 inhibitor (TFMI-2) variants are potent and selective LP inhibitors in both human and rat serum. Directed evolution of the first Kunitz domain of TFPI1 had already yielded the potent kallikrein inhibitor, Kalbitor® (ecallantide), which is an FDA-approved drug to treat acute attacks of hereditary angioedema. Like hereditary angioedema, acute IRI is also related to the uncontrolled activation of a specific plasma serine proteinase. Therefore, TFMI-2 variants are promising lead molecules for drug development against IRI.

Overlapping Specificity of Duplicated Human Pancreatic Elastase 3 Isoforms and Archetypal Porcine Elastase 1 Provides Clues to Evolution of Digestive Enzymes.

Chymotrypsin-like elastases (CELAs) are pancreatic serine proteinases that digest dietary proteins. CELAs are typically expressed in multiple isoforms that can vary among different species. The human pancreas does not express CELA1 but secretes two CELA3 isoforms, CELA3A and CELA3B. The reasons for the CELA3 duplication and the substrate preferences of the duplicated isoforms are unclear. Here, we tested whether CELA3A and CELA3B evolved unique substrate specificities to compensate for the loss of CELA1. We constructed a phage library displaying variants of the substrate-like Schistocerca gregaria proteinase inhibitor 2 (SGPI-2) to select reversible high affinity inhibitors of human CELA3A, CELA3B, and porcine CELA1. Based on the reactive loop sequences of the phage display-selected inhibitors, we recombinantly expressed and purified 12 SGPI-2 variants and determined their binding affinities. We found that the primary specificity of CELA3A, CELA3B, and CELA1 was similar; all preferred aliphatic side chains at the so-called P1 position, the amino acid residue located directly N-terminal to the scissile peptide bond. P1 Met was an interesting exception that was preferred by CELA1 but weakly recognized by the CELA3 isoforms. The extended substrate specificity of CELA3A and CELA3B was comparable, whereas CELA1 exhibited unique interactions at several subsites. These observations indicated that the CELA1 and CELA3 paralogs have some different but also overlapping specificities and that the duplicated CELA3A and CELA3B isoforms did not evolve distinct substrate preferences. Thus, increased gene dosage rather than specificity divergence of the CELA3 isoforms may compensate for the loss of CELA1 digestive activity in the human pancreas.

Structural determinants governing S100A4-induced isoform-selective disassembly of nonmuscle myosin II filaments.

The Ca(2+) -binding protein S100A4 interacts with the C terminus of nonmuscle myosin IIA (NMIIA) causing filament disassembly, which is correlated with an increased metastatic potential of tumor cells. Despite high sequence similarity of the three NMII isoforms, S100A4 discriminates against binding to NMIIB. We searched for structural determinants of this selectivity. Based on paralog scanning using phage display, we identified a single position as major determinant of isoform selectivity. Reciprocal single amino acid replacements showed that at position 1907 (NMIIA numbering), the NMIIA/NMIIC-specific alanine provides about 60-fold higher affinity than the NMIIB-specific asparagine. The structural background of this can be explained in part by a communication between the two consecutive α-helical binding segments. This communication is completely abolished by the Ala-to-Asn substitution. Mutual swapping of the disordered tailpieces only slightly affects the affinity of the NMII chimeras. Interestingly, we found that the tailpiece and position 1907 act in a nonadditive fashion. Finally, we also found that the higher stability of the C-terminal coiled-coil region of NMIIB also discriminates against interaction with S100A4. Our results clearly show that the isoform-selective binding of S100A4 is determined at multiple levels in the structure of the three NMII isoforms and the corresponding functional elements of NMII act synergistically with one another resulting in a complex interaction network. The experimental and in silico results suggest two divergent evolutionary pathways: NMIIA and NMIIB evolved to possess S100A4-dependent and -independent regulations, respectively.

Quantitative characterization of the activation steps of mannan-binding lectin (MBL)-associated serine proteases (MASPs) points to the central role of MASP-1 in the initiation of the complement lectin pathway.

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.

DYNLL/LC8: a light chain subunit of the dynein motor complex and beyond.

The LC8 family members of dynein light chains (DYNLL1 and DYNLL2 in vertebrates) are highly conserved ubiquitous eukaryotic homodimer proteins that interact, besides dynein and myosin 5a motor proteins, with a large (and still incomplete) number of proteins involved in diverse biological functions. Despite an earlier suggestion that LC8 light chains function as cargo adapters of the above molecular motors, they are now recognized as regulatory hub proteins that interact with short linear motifs located in intrinsically disordered protein segments. The most prominent LC8 function is to promote dimerization of their binding partners that are often scaffold proteins of various complexes, including the intermediate chains of the dynein motor complex. Structural and functional aspects of this intriguing hub protein will be highlighted in this minireview.

High affinity small protein inhibitors of human chymotrypsin C (CTRC) selected by phage display reveal unusual preference for P4' acidic residues.

Human chymotrypsin C (CTRC) is a pancreatic protease that participates in the regulation of intestinal digestive enzyme activity. Other chymotrypsins and elastases are inactive on the regulatory sites cleaved by CTRC, suggesting that CTRC recognizes unique sequence patterns. To characterize the molecular determinants underlying CTRC specificity, we selected high affinity substrate-like small protein inhibitors against CTRC from a phage library displaying variants of SGPI-2, a natural chymotrypsin inhibitor from Schistocerca gregaria. On the basis of the sequence pattern selected, we designed eight inhibitor variants in which amino acid residues in the reactive loop at P1 (Met or Leu), P2' (Leu or Asp), and P4' (Glu, Asp, or Ala) were varied. Binding experiments with CTRC revealed that (i) inhibitors with Leu at P1 bind 10-fold stronger than those with P1 Met; (ii) Asp at P2' (versus Leu) decreases affinity but increases selectivity, and (iii) Glu or Asp at P4' (versus Ala) increase affinity 10-fold. The highest affinity SGPI-2 variant (K(D) 20 pm) bound to CTRC 575-fold tighter than the parent molecule. The most selective inhibitor variant exhibited a K(D) of 110 pm and a selectivity ranging from 225- to 112,664-fold against other human chymotrypsins and elastases. Homology modeling and mutagenesis identified a cluster of basic amino acid residues (Lys(51), Arg(56), and Arg(80)) on the surface of human CTRC that interact with the P4' acidic residue of the inhibitor. The acidic preference of CTRC at P4' is unique among pancreatic proteases and might contribute to the high specificity of CTRC-mediated digestive enzyme regulation.

Reversible heat-induced dissociation of ß2-microglobulin amyloid fibrils.

Recent progress in the field of amyloid research indicates that the classical view of amyloid fibrils, being irreversibly formed highly stable structures resistant to perturbating conditions and proteolytic digestion, is getting more complex. We studied the thermal stability and heat-induced depolymerization of amyloid fibrils of ß(2)-microglobulin (ß2m), a protein responsible for dialysis-related amyloidosis. We found that freshly polymerized ß2m fibrils at 0.1-0.3 mg/mL concentration completely dissociated to monomers upon 10 min incubation at 99 °C. Fibril depolymerization was followed by thioflavin-T fluorescence and circular dichroism spectroscopy at various temperatures. Dissociation of ß2m fibrils was found to be a reversible and dynamic process reaching equilibrium between fibrils and monomers within minutes. Repolymerization experiments revealed that the number of extendable fibril ends increased significantly upon incubation at elevated temperatures suggesting that the mechanism of fibril unfolding involves two distinct processes: (1) dissociation of monomers from the fibril ends and (2) the breakage of fibrils. The breakage of fibrils may be an important in vivo factor multiplying the number of fibril nuclei and thus affecting the onset and progress of disease. We investigated the effects of some additives and different factors on the stability of amyloid fibrils. Sample aging increased the thermal stability of ß2m fibril solution. 0.5 mM SDS completely prevented ß2m fibrils from dissociation up to the applied highest temperature of 99 °C. The generality of our findings was proved on fibrils of K3 peptide and α-synuclein. Our simple method may also be beneficial for screening and developing amyloid-active compounds for therapeutic purposes.

Mechanism of lysophosphatidic acid-induced amyloid fibril formation of beta(2)-microglobulin in vitro under physiological conditions.

Beta(2)-microglobulin- (beta2m-) based fibril deposition is the key symptom in dialysis-related amyloidosis. beta2m readily forms amyloid fibrils in vitro at pH 2.5. However, it is not well understood which factors promote this process in vivo, because beta2m cannot polymerize at neutral pH without additives even at elevated concentration. Here we show that lysophosphatidic acid (LPA), an in vivo occurring lysophospholipid mediator, promotes amyloid formation under physiological conditions through a complex mechanism. In the presence of LPA, at and above its critical micelle concentration, native beta2m became sensitive to limited proteolytic digestion, indicating increased conformational flexibility. Isothermal titration calorimetry indicates that beta2m exhibits high affinity for LPA. Fluorescence and CD spectroscopy, as well as calorimetry, showed that LPA destabilizes the structure of monomeric beta2m inducing a partially unfolded form. This intermediate is capable of fibril extension in a nucleation-dependent manner. Our findings also indicate that the molecular organization of fibrils formed under physiological conditions differs from that of fibrils formed at pH 2.5. Fibrils grown in the presence of LPA depolymerize very slowly in the absence of LPA; moreover, LPA stabilizes the fibrils even below its critical micelle concentration. Neither the amyloidogenic nor the fibril-stabilizing effects of LPA were mimicked by its structural and functional lysophospholipid analogues, showing its selectivity. On the basis of our findings and the observed increase in blood LPA levels in dialysis patients, we suggest that the interaction of LPA with beta2m might contribute to the pathomechanism of dialysis-related amyloidosis.

When the surface tells what lies beneath: combinatorial phage-display mutagenesis reveals complex networks of surface-core interactions in the pacifastin protease inhibitor family.

Pacifastin protease inhibitors are small cysteine-rich motifs of approximately 35 residues that were discovered in arthropods. The family is divided into two related groups on the basis of the composition of their minimalist inner core. In group I, the core is governed by a Lys10-Trp26 interaction, while in group II it is organized around Phe10. Group I inhibitors exhibit intriguing taxon specificity: potent arthropod-trypsin inhibitors from this group are almost inactive against vertebrate enzymes. The group I member SGPI-1 and the group II member SGPI-2 are extensively studied inhibitors. SGPI-1 is taxon-selective, while SGPI-2 is not. Individual mutations failed to explain the causes underlying this difference. We deciphered this phenomenon using comprehensive combinatorial mutagenesis and phage display. We produced a complete chimeric SGPI-1 / SGPI-2 inhibitor-phage library, in which the two sequences were shuffled at the highest possible resolution of individual residues. The library was selected for binding to bovine trypsin and crayfish trypsin. Sequence analysis of the selectants revealed that taxon specificity is due to an intra-molecular functional coupling between a surface loop and the Lys10-Trp26 core. Five SGPI-2 surface residues transplanted into SGPI-1 resulted in a variant that retained the "taxon-specific" core, but potently inhibited both vertebrate and arthropod enzymes. An additional rational point mutation resulted in a picomolar inhibitor of both trypsins. Our results challenge the generally accepted view that surface residues are the exclusive source of selectivity for canonical inhibitors. Moreover, we provide important insights into general principles underlying the structure-function properties of small disulfide-rich polypeptides, molecules that exist at the borderline between peptides and proteins.

Mutant rat trypsin selectively cleaves tyrosyl peptide bonds.

A double mutant of rat trypsinogen (Asp189Ser, DeltaAsp223) was constructed by site-directed mutagenesis. The recombinant protein was produced in Escherichia coli under the control of a periplasmic expression vector. The purified and enterokinase-activated enzyme was characterized by synthetic fluorogenic tetrapeptide and natural polypeptide substrates and by a recently developed method. In case of this latter method the specificity profile of the enzyme was examined by simultaneous digestion of a mixture of oligopeptide substrates each differing only at the P(1) site residue, and the results were analyzed by high-performance liquid chromatography. All these assays unanimously demonstrated that the recombinant proteinase lacks trypsin-like activity but acquired a rather unique selectivity: it preferentially hydrolyses peptide bonds C-terminal to tyrosyl residues. This narrow specificity should be useful in peptide-analytical applications such as sequence-specific fragmentation of large proteins prior to sequencing.

Reexamination of the recognition preference of the specificity pocket of the Abl SH3 domain.

Src homology-3 (SH3) domains mediate important protein-protein interactions in a variety of normal and pathological cellular processes, thus providing an attractive target for the selective interference of SH3-dependent signaling events that govern these processes. Most SH3 domains recognize proline-rich peptides with low affinity and poor selectivity, and the goal to design potent and specific ligands for various SH3 domains remains elusive. Better understanding of the molecular basis for SH3 domain recognition is needed in order to design such ligands with potency and specificity. In this report, we seek to define a clear recognition preference of the specificity pocket of the Abl SH3 domain using targeted synthetic peptide libraries. High-resolution affinity panning coupled with mass spectrometric readout allows for quick identification of Trp as the preferred fourth residue in the decapeptide ligand APTWSPPPPP, which binds to Abl SH3 four times stronger than does the decapeptide containing Tyr or Phe in the fourth position. This finding is in contrast to several reports that Tyr is the only residue selected from phage displayed peptide libraries that interacts with the specificity pocket of Abl SH3. This simple, unbiased approach can fine-tune the affinity and selectivity of both natural and unnatural SH3 ligands whose consensus binding sequence has been pre-defined by combinatorial library methods.

The first semi-synthetic serine protease made by native chemical ligation.

Selective incorporation of non-natural amino acid residues into proteins is a powerful approach to delineate structure-function relationships. Although many methodologies are available for chemistry-based protein engineering, more facile methods are needed to make this approach suitable for routine laboratory practice. Here, we describe a new strategy and provide a proof of concept for engineering semi-synthetic proteins. We chose a serine protease Streptomyces griseus trypsin (SGT) for this study to show that it is possible to efficiently couple a synthetic peptide containing a catalytically critical residue to a recombinant fragment containing the other active site residues. The 223-residue hybrid SGT molecule was prepared by fusing a chemically synthesized N-terminal peptide to a large C-terminal fragment of recombinant origin using native chemical ligation. This C-terminal polypeptide was produced from full-length SGT by cyanogen bromide cleavage at a genetically engineered Met57 position. This semi-synthetic hybrid trypsin is fully active, showing kinetics identical to the wild-type enzyme. Thus, we believe that it is an ideal model enzyme for studying the catalytic mechanisms of serine proteases by providing a straightforward approach to incorporate non-natural amino acids in the N-terminal region of the protein. In particular, this strategy will allow for replacement of the catalytic His57 residue and the buried N-terminus, which is thought to help align the active site, with synthetic analogs. Our approach relies on readily available recombinant proteins and small synthetic peptides, thus having general applications in chemical engineering of large proteins where the N-terminal region is the focal interest.