Structural and functional insights into the C-terminal signal domain of the Bacteroidetes type-IX secretion system.

Gram-negative bacteria from the Bacteroidota phylum possess a type-IX secretion system (T9SS) for protein secretion, which requires cargoes to have a C-terminal domain (CTD). Structurally analysed CTDs are from Porphyromonas gingivalis proteins RgpB, HBP35, PorU and PorZ, which share a compact immunoglobulin-like antiparallel 3+4 ß-sandwich (ß1-ß7). This architecture is essential as a P. gingivalis strain with a single-point mutant of RgpB disrupting the interaction of the CTD with its preceding domain prevented secretion of the protein. Next, we identified the C-terminus ('motif C-t.') and the loop connecting strands ß3 and ß4 ('motif Lß3ß4') as conserved. We generated two strains with insertion and replacement mutants of PorU, as well as three strains with ablation and point mutants of RgpB, which revealed both motifs to be relevant for T9SS function. Furthermore, we determined the crystal structure of the CTD of mirolase, a cargo of the Tannerella forsythia T9SS, which shares the same general topology as in Porphyromonas CTDs. However, motif Lß3ß4 was not conserved. Consistently, P. gingivalis could not properly secrete a chimaeric protein with the CTD of peptidylarginine deiminase replaced with this foreign CTD. Thus, the incompatibility of the CTDs between these species prevents potential interference between their T9SSs.

Frozen fresh blood plasma preserves the functionality of native human α2-macroglobulin.

Human α2-macroglobulin (hα2M) is a large homotetrameric protein involved in the broad inhibition of endopeptidases. Following cleavage within a bait region, hα2M undergoes stepwise transitions from its native, expanded, highly flexible, active conformation to an induced, compact, triggered conformation. As a consequence, the peptidase is entrapped by an irreversible Venus flytrap mechanism. Given the importance of hα2M, biochemical studies galore over more than seven decades have attempted to ascertain its role, typically using authentic hα2M purified from frozen and non-frozen fresh blood plasma, and even outdated plasma. However, hα2M is sensitive once isolated and purified, and becomes heterogeneous during storage and/or freezing, raising concerns about the functional competence of frozen plasma-derived hα2M. We therefore used a combination of native and sodium dodecylsulfate polyacrylamide gel electrophoresis, affinity and ion-exchange chromatography, multi-angle laser light scattering after size-exclusion chromatography, free cysteine quantification, and peptidase inhibition assays with endopeptidases of two catalytic classes and three protein substrates, to characterize the biochemical and biophysical properties of hα2M purified ad hoc either from fresh plasma or frozen fresh plasma after thawing. We found no differences in the molecular or functional properties of the preparations, indicating that protective components in plasma maintain native hα2M in a functionally competent state despite freezing.

Structural insights into latency of the metallopeptidase ulilysin (lysargiNase) and its unexpected inhibition by a sulfonyl-fluoride inhibitor of serine peptidases.

Peptidases are regulated by latency and inhibitors, as well as compatibilization and cofactors. Ulilysin from Methanosarcina acetivorans, also called lysargiNase, is an archaeal metallopeptidase (MP) that is biosynthesized as a zymogen with a 60-residue N-terminal prosegment (PS). In the presence of calcium, it self-activates to yield the mature enzyme, which specifically cleaves before basic residues and thus complements trypsin in proteomics workflows. Here, we obtained a low-resolution crystal structure of proulilysin, in which 28 protomers arranged as 14 dimers form a continuous double helix of 544 Å pitch that parallels cell axis b of the crystal. The PS includes two α-helices and obstructs the active-site cleft of the catalytic domain (CD) by traversing it in the opposite orientation of a substrate, and a cysteine blocks the catalytic zinc according to a "cysteine-switch mechanism". Moreover, the PS interacts through its first helix with an "S-loop" of the CD, which acts as an "activation segment" that lacks one of two essential calcium cations. Upon PS removal during maturation, the S-loop adopts its competent conformation and binds the second calcium ion. Next, we found that in addition to general MP inhibitors, ulilysin was competitively and reversibly inhibited by 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; Ki = 4 µM). This is a compound that normally forms an irreversible covalent complex with serine peptidases but does not inhibit MPs. A high-resolution crystal structure of the complex revealed that the inhibitor penetrates the specificity pocket of ulilysin. A primary amine of the inhibitor salt-bridges an aspartate at the pocket bottom, thus mimicking the basic side chain of substrates. In contrast, the sulfonyl fluoride warhead is not involved and the catalytic zinc ion is freely accessible. Thus, the usage of inhibitor cocktails of peptidases, which typically contain AEBSF at ∼25-fold higher concentrations than the determined Ki, should be avoided when working with ulilysin. Finally, the structure of the complex, which occurred as a crystallographic dimer recurring in previous mature ulilysin structures, unveiled an N-terminal product fragment that delineated the non-primed side of the cleft. These results complement prior structures of ulilysin with primed-side product fragments and inhibitors.

A unique network of attack, defence and competence on the outer membrane of the periodontitis pathogen Tannerella forsythia.

Periodontopathogenic Tannerella forsythia uniquely secretes six peptidases of disparate catalytic classes and families that operate as virulence factors during infection of the gums, the KLIKK-peptidases. Their coding genes are immediately downstream of novel ORFs encoding the 98-132 residue potempins (Pot) A, B1, B2, C, D and E. These are outer-membrane-anchored lipoproteins that specifically and potently inhibit the respective downstream peptidase through stable complexes that protect the outer membrane of T. forsythia, as shown in vivo. Remarkably, PotA also contributes to bacterial fitness in vivo and specifically inhibits matrix metallopeptidase (MMP) 12, a major defence component of oral macrophages, thus featuring a novel and highly-specific physiological MMP inhibitor. Information from 11 structures and high-confidence homology models showed that the potempins are distinct ß-barrels with either a five-stranded OB-fold (PotA, PotC and PotD) or an eight-stranded up-and-down fold (PotE, PotB1 and PotB2), which are novel for peptidase inhibitors. Particular loops insert like wedges into the active-site cleft of the genetically-linked peptidases to specifically block them either via a new "bilobal" or the classic "standard" mechanism of inhibition. These results discover a unique, tightly-regulated proteolytic armamentarium for virulence and competence, the KLIKK-peptidase/potempin system.

Zymogenic latency in an ∼250-million-year-old astacin metallopeptidase.

The horseshoe crab Limulus polyphemus is one of few extant Limulus species, which date back to ∼250 million years ago under the conservation of a common Bauplan documented by fossil records. It possesses the only proteolytic blood-coagulation and innate immunity system outside vertebrates and is a model organism for the study of the evolution and function of peptidases. The astacins are a family of metallopeptidases that share a central ∼200-residue catalytic domain (CD), which is found in >1000 species across holozoans and, sporadically, bacteria. Here, the zymogen of an astacin from L. polyphemus was crystallized and its structure was solved. A 34-residue, mostly unstructured pro-peptide (PP) traverses, and thus blocks, the active-site cleft of the CD in the opposite direction to a substrate. A central `PP motif' (F35-E-G-D-I39) adopts a loop structure which positions Asp38 to bind the catalytic metal, replacing the solvent molecule required for catalysis in the mature enzyme according to an `aspartate-switch' mechanism. Maturation cleavage of the PP liberates the cleft and causes the rearrangement of an `activation segment'. Moreover, the mature N-terminus is repositioned to penetrate the CD moiety and is anchored to a buried `family-specific' glutamate. Overall, this mechanism of latency is reminiscent of that of the other three astacins with known zymogenic and mature structures, namely crayfish astacin, human meprin ß and bacterial myroilysin, but each shows specific structural characteristics. Remarkably, myroilysin lacks the PP motif and employs a cysteine instead of the aspartate to block the catalytic metal.

De novo design of immunoglobulin-like domains.

Antibodies, and antibody derivatives such as nanobodies, contain immunoglobulin-like (Ig) ß-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here, we develop design rules for the central feature of the Ig fold architecture-the non-local cross-ß structure connecting the two ß-sheets-and use these to design highly stable Ig domains de novo, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of antibody-like scaffolds with tailored structures and superior biophysical properties.

Molecular and in vivo studies of a glutamate-class prolyl-endopeptidase for coeliac disease therapy.

The digestion of gluten generates toxic peptides, among which a highly immunogenic proline-rich 33-mer from wheat α-gliadin, that trigger coeliac disease. Neprosin from the pitcher plant is a reported prolyl endopeptidase. Here, we produce recombinant neprosin and its mutants, and find that full-length neprosin is a zymogen, which is self-activated at gastric pH by the release of an all-ß pro-domain via a pH-switch mechanism featuring a lysine plug. The catalytic domain is an atypical 7+8-stranded ß-sandwich with an extended active-site cleft containing an unprecedented pair of catalytic glutamates. Neprosin efficiently degrades both gliadin and the 33-mer in vitro under gastric conditions and is reversibly inactivated at pH > 5. Moreover, co-administration of gliadin and the neprosin zymogen at the ratio 500:1 reduces the abundance of the 33-mer in the small intestine of mice by up to 90%. Neprosin therefore founds a family of eukaryotic glutamate endopeptidases that fulfils requisites for a therapeutic glutenase.

Cryo-EM structures show the mechanistic basis of pan-peptidase inhibition by human α2-macroglobulin.

Human α2-macroglobulin (hα2M) is a multidomain protein with a plethora of essential functions, including transport of signaling molecules and endopeptidase inhibition in innate immunity. Here, we dissected the molecular mechanism of the inhibitory function of the ∼720-kDa hα2M tetramer through eight cryo­electron microscopy (cryo-EM) structures of complexes from human plasma. In the native complex, the hα2M subunits are organized in two flexible modules in expanded conformation, which enclose a highly porous cavity in which the proteolytic activity of circulating plasma proteins is tested. Cleavage of bait regions exposed inside the cavity triggers rearrangement to a compact conformation, which closes openings and entraps the prey proteinase. After the expanded-to-compact transition, which occurs independently in the four subunits, the reactive thioester bond triggers covalent linking of the proteinase, and the receptor-binding domain is exposed on the tetramer surface for receptor-mediated clearance from circulation. These results depict the molecular mechanism of a unique suicidal inhibitory trap.

An engineered protein-based submicromolar competitive inhibitor of the Staphylococcus aureus virulence factor aureolysin.

Aureolysin, a secreted metallopeptidase (MP) from the thermolysin family, functions as a major virulence factor in Staphylococcus aureus. No specific aureolysin inhibitors have yet been described, making this an important target for the development of novel antimicrobial drugs in times of rampant antibiotic resistance. Although small-molecule inhibitors are currently more common in the clinic, therapeutic proteins and peptides (TPs) are favourable due to their high selectivity, which reduces off-target toxicity and allows dosage tuning. The greater wax moth Galleria mellonella produces a unique defensive protein known as the insect metallopeptidase inhibitor (IMPI), which selectively inhibits some thermolysins from pathogenic bacteria. We determined the ability of IMPI to inhibit aureolysin in vitro and used crystal structures to ascertain its mechanism of action. This revealed that IMPI uses the "standard mechanism", which has been poorly characterised for MPs in general. Accordingly, we designed a cohort of 12 single and multiple point mutants, the best of which (I57F) inhibited aureolysin with an estimated inhibition constant (K i) of 346 nM. Given that animals lack thermolysins, our strategy may facilitate the development of safe TPs against staphylococcal infections, including strains resistant to conventional antibiotics.

Crystal structure report of the ImmR transcriptional regulator DNA-binding domain of the Bacillus subtilis ICEBs1 transposon.

Bacillus subtilis is a commensal member of the human oral and gut microbiomes, which can become infectious to immunocompromised patients. It possesses a conjugative transposon, ICEBs1, which includes > 20 genes and can be passed by horizontal gene transfer to other bacteria, including pathogenic Bacillus anthracis and Listeria monocytogenes. ICEBs1 is regulated by the ImmR/ImmA tandem, which are a transcriptional repressor that constitutively blocks transcription and a metallopeptidase that acts as anti-repressor and inactivates ImmR by proteolytic cleavage. We here report the production and purification of 127-residue ImmR from ICEBs1 and the crystal structure of its DNA-binding domain. It features a five-helix bundle centred on a helix-turn-helix motif potentially binding the major grove of double-stranded target DNA. ImmR shows structural and mechanistic similarity with the B. subtilis SinR repressor, which is engaged in sporulation inhibition.

Structural and evolutionary insights into astacin metallopeptidases.

The astacins are a family of metallopeptidases (MPs) that has been extensively described from animals. They are multidomain extracellular proteins, which have a conserved core architecture encompassing a signal peptide for secretion, a prodomain or prosegment and a zinc-dependent catalytic domain (CD). This constellation is found in the archetypal name-giving digestive enzyme astacin from the European crayfish Astacus astacus. Astacin catalytic domains span ∼200 residues and consist of two subdomains that flank an extended active-site cleft. They share several structural elements including a long zinc-binding consensus sequence (HEXXHXXGXXH) immediately followed by an EXXRXDRD motif, which features a family-specific glutamate. In addition, a downstream SIMHY-motif encompasses a "Met-turn" methionine and a zinc-binding tyrosine. The overall architecture and some structural features of astacin catalytic domains match those of other more distantly related MPs, which together constitute the metzincin clan of metallopeptidases. We further analysed the structures of PRO-, MAM, TRAF, CUB and EGF-like domains, and described their essential molecular determinants. In addition, we investigated the distribution of astacins across kingdoms and their phylogenetic origin. Through extensive sequence searches we found astacin CDs in > 25,000 sequences down the tree of life from humans beyond Metazoa, including Choanoflagellata, Filasterea and Ichtyosporea. We also found < 400 sequences scattered across non-holozoan eukaryotes including some fungi and one virus, as well as in selected taxa of archaea and bacteria that are pathogens or colonizers of animal hosts, but not in plants. Overall, we propose that astacins originate in the root of Holozoa consistent with Darwinian descent and that the latter genes might be the result of horizontal gene transfer from holozoan donors.

Intermolecular latency regulates the essential C-terminal signal peptidase and sortase of the Porphyromonas gingivalis type-IX secretion system.

Porphyromonas gingivalis is a keystone pathogen of the human dysbiotic oral microbiome that causes severe periodontitis. It employs a type-IX secretion system (T9SS) to shuttle proteins across the outer membrane (OM) for virulence. Uniquely, T9SS cargoes carry a C-terminal domain (CTD) as a secretion signal, which is cleaved and replaced with anionic lipopolysaccharide by transpeptidation for extracellular anchorage to the OM. Both reactions are carried out by PorU, the only known dual-function, C-terminal signal peptidase and sortase. PorU is itself secreted by the T9SS, but its CTD is not removed; instead, intact PorU combines with PorQ, PorV, and PorZ in the OM-inserted "attachment complex." Herein, we revealed that PorU transits between active monomers and latent dimers and solved the crystal structure of the ∼260-kDa dimer. PorU has an elongated shape ∼130 Å in length and consists of seven domains. The first three form an intertwined N-terminal cluster likely engaged in substrate binding. They are followed by a gingipain-type catalytic domain (CD), two immunoglobulin-like domains (IGL), and the CTD. In the first IGL, a long "latency ß-hairpin" protrudes ∼30 Å from the surface to form an intermolecular ß-barrel with ß-strands from the symmetric CD, which is in a latent conformation. Homology modeling of the competent CD followed by in vivo validation through a cohort of mutant strains revealed that PorU is transported and functions as a monomer through a C690/H657 catalytic dyad. Thus, dimerization is an intermolecular mechanism for PorU regulation to prevent untimely activity until joining the attachment complex.

The crystal structure of a 250-kDa heterotetrameric particle explains inhibition of sheddase meprin ß by endogenous fetuin-B.

Meprin ß (Mß) is a multidomain type-I membrane metallopeptidase that sheds membrane-anchored substrates, releasing their soluble forms. Fetuin-B (FB) is its only known endogenous protein inhibitor. Herein, we analyzed the interaction between the ectodomain of Mß (MßΔC) and FB, which stabilizes the enzyme and inhibits it with subnanomolar affinity. The MßΔC:FB crystal structure reveals a ∼250-kDa, ∼160-Å polyglycosylated heterotetrameric particle with a remarkable glycan structure. Two FB moieties insert like wedges through a "CPDCP trunk" and two hairpins into the respective peptidase catalytic domains, blocking the catalytic zinc ions through an "aspartate switch" mechanism. Uniquely, the active site clefts are obstructed from subsites S4 to S10', but S1 and S1' are spared, which prevents cleavage. Modeling of full-length Mß reveals an EGF-like domain between MßΔC and the transmembrane segment that likely serves as a hinge to transit between membrane-distal and membrane-proximal conformations for inhibition and catalysis, respectively.

An Integrative Structural Biology Analysis of Von Willebrand Factor Binding and Processing by ADAMTS-13 in Solution.

Von Willebrand Factor (vWF), a 300-kDa plasma protein key to homeostasis, is cleaved at a single site by multi-domain metallopeptidase ADAMTS-13. vWF is the only known substrate of this peptidase, which circulates in a latent form and becomes allosterically activated by substrate binding. Herein, we characterised the complex formed by a competent peptidase construct (AD13-MDTCS) comprising metallopeptidase (M), disintegrin-like (D), thrombospondin (T), cysteine-rich (C), and spacer (S) domains, with a 73-residue functionally relevant vWF-peptide, using nine complementary techniques. Pull-down assays, gel electrophoresis, and surface plasmon resonance revealed tight binding with sub-micromolar affinity. Cross-linking mass spectrometry with four reagents showed that, within the peptidase, domain D approaches M, C, and S. S is positioned close to M and C, and the peptide contacts all domains. Hydrogen/deuterium exchange mass spectrometry revealed strong and weak protection for C/D and M/S, respectively. Structural analysis by multi-angle laser light scattering and small-angle X-ray scattering in solution revealed that the enzyme adopted highly flexible unbound, latent structures and peptide-bound, active structures that differed from the AD13-MDTCS crystal structure. Moreover, the peptide behaved like a self-avoiding random chain. We integrated the results with computational approaches, derived an ensemble of structures that collectively satisfied all experimental restraints, and discussed the functional implications. The interaction conforms to a 'fuzzy complex' that follows a 'dynamic zipper' mechanism involving numerous reversible, weak but additive interactions that result in strong binding and cleavage. Our findings contribute to illuminating the biochemistry of the vWF:ADAMTS-13 axis.

PorZ, an Essential Component of the Type IX Secretion System of Porphyromonas gingivalis, Delivers Anionic Lipopolysaccharide to the PorU Sortase for Transpeptidase Processing of T9SS Cargo Proteins.

Cargo proteins of the type IX secretion system (T9SS) in human pathogens from the Bacteroidetes phylum invariably possess a conserved C-terminal domain (CTD) that functions as a signal for outer membrane (OM) translocation. In Porphyromonas gingivalis, the CTD of cargos is cleaved off after translocation, and anionic lipopolysaccharide (A-LPS) is attached. This transpeptidase reaction anchors secreted proteins to the OM. PorZ, a cell surface-associated protein, is an essential component of the T9SS whose function was previously unknown. We recently solved the crystal structure of PorZ and found that it consists of two ß-propeller moieties, followed by a CTD. In this study, we performed structure-based modeling, suggesting that PorZ is a carbohydrate-binding protein. Indeed, we found that recombinant PorZ specifically binds A-LPS in vitro Binding was blocked by monoclonal antibodies that specifically react with a phosphorylated branched mannan in the anionic polysaccharide (A-PS) component of A-LPS, but not with the core oligosaccharide or the lipid A endotoxin. Examination of A-LPS derived from a cohort of mutants producing various truncations of A-PS confirmed that the phosphorylated branched mannan is indeed the PorZ ligand. Moreover, purified recombinant PorZ interacted with the PorU sortase in an A-LPS-dependent manner. This interaction on the cell surface is crucial for the function of the "attachment complex" composed of PorU, PorZ, and the integral OM ß-barrel proteins PorV and PorQ, which is involved in posttranslational modification and retention of T9SS cargos on the bacterial surface.IMPORTANCE Bacteria have evolved multiple systems to transport effector proteins to their surface or into the surrounding milieu. These proteins have a wide range of functions, including attachment, motility, nutrient acquisition, and toxicity in the host. Porphyromonas gingivalis, the human pathogen responsible for severe gum diseases (periodontitis), uses a recently characterized type IX secretion system (T9SS) to translocate and anchor secreted virulence effectors to the cell surface. Anchorage is facilitated by sortase, an enzyme that covalently attaches T9SS cargo proteins to a unique anionic lipopolysaccharide (A-LPS) moiety of P. gingivalis Here, we show that the T9SS component PorZ interacts with sortase and specifically binds A-LPS. Binding is mediated by a phosphorylated branched mannan repeat in A-LPS polysaccharide. A-LPS-bound PorZ interacts with sortase with significantly higher affinity, facilitating modification of cargo proteins by the cell surface attachment complex of the T9SS.

Analysis of the inhibiting activity of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) on matrix metalloproteinases.

Matrix metalloproteinases (MMPs) occur in 23 human paralogues with key functions in physiology, and their activity is controlled by protein inhibitors. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), which is essential for embryogenesis and tumour suppression, has been reported to inhibit MMPs. Here, we developed eukaryotic and bacterial expression systems for different RECK variants and analysed their inhibitory capacity against representative MMPs in vitro. We could not detect any significant inhibition. Instead, we found that partially purified RECK from the conditioned medium of transfected Expi293F cells but not that of ExpiCHO-S or Drosophila Schneider cells contained a contaminant with proteolytic activity. The contaminant was removed through treatment with a small-molecule serine peptidase inhibitor and additional chromatographic purification. A tantamount contaminant was further detected in an equivalent expression system of the N-terminal fragment of the proteoglycan testican 3, but not in those of two other proteins. These results indicate that previous reports of inhibitory activity of recombinant RECK on MMPs, which were performed with partially purified samples, were probably masked by a coeluting contaminant present in the supernatant of HEK293-derived cells. Thus, RECK is probably not a direct inhibitor of MMP catalytic activity but may still regulate MMPs through other mechanisms.

Structure-based mechanism of cysteine-switch latency and of catalysis by pappalysin-family metallopeptidases.

Tannerella forsythia is an oral dysbiotic periodontopathogen involved in severe human periodontal disease. As part of its virulence factor armamentarium, at the site of colonization it secretes mirolysin, a metallopeptidase of the unicellular pappalysin family, as a zymogen that is proteolytically auto-activated extracellularly at the Ser54-Arg55 bond. Crystal structures of the catalytically impaired promirolysin point mutant E225A at 1.4 and 1.6 Šrevealed that latency is exerted by an N-terminal 34-residue pro-segment that shields the front surface of the 274-residue catalytic domain, thus preventing substrate access. The catalytic domain conforms to the metzincin clan of metallopeptidases and contains a double calcium site, which acts as a calcium switch for activity. The pro-segment traverses the active-site cleft in the opposite direction to the substrate, which precludes its cleavage. It is anchored to the mature enzyme through residue Arg21, which intrudes into the specificity pocket in cleft sub-site S1'. Moreover, residue Cys23 within a conserved cysteine-glycine motif blocks the catalytic zinc ion by a cysteine-switch mechanism, first described for mammalian matrix metallopeptidases. In addition, a 1.5 Šstructure was obtained for a complex of mature mirolysin and a tetradecapeptide, which filled the cleft from sub-site S1' to S6'. A citrate molecule in S1 completed a product-complex mimic that unveiled the mechanism of substrate binding and cleavage by mirolysin, the catalytic domain of which was already preformed in the zymogen. These results, including a preference for cleavage before basic residues, are likely to be valid for other unicellular pappalysins derived from archaea, bacteria, cyanobacteria, algae and fungi, including archetypal ulilysin from Methanosarcina acetivorans. They may further apply, at least in part, to the multi-domain orthologues of higher organisms.

The C-terminal region of human plasma fetuin-B is dispensable for the raised-elephant-trunk mechanism of inhibition of astacin metallopeptidases.

Human fetuin-B plays a key physiological role in human fertility through its inhibitory action on ovastacin, a member of the astacin family of metallopeptidases. The inhibitor consists of tandem cystatin-like domains (CY1 and CY2), which are connected by a linker containing a "CPDCP-trunk" and followed by a C-terminal region (CTR) void of regular secondary structure. Here, we solved the crystal structure of the complex of the inhibitor with archetypal astacin from crayfish, which is a useful model of human ovastacin. Two hairpins from CY2, the linker, and the tip of the "legumain-binding loop" of CY1 inhibit crayfish astacin following the "raised-elephant-trunk mechanism" recently reported for mouse fetuin-B. This inhibition is exerted by blocking active-site cleft sub-sites upstream and downstream of the catalytic zinc ion, but not those flanking the scissile bond. However, contrary to the mouse complex, which was obtained with fetuin-B nicked at a single site but otherwise intact, most of the CTR was proteolytically removed during crystallization of the human complex. Moreover, the two complexes present in the crystallographic asymmetric unit diverged in the relative arrangement of CY1 and CY2, while the two complexes found for the mouse complex crystal structure were equivalent. Biochemical studies in vitro confirmed the differential cleavage susceptibility of human and mouse fetuin-B in front of crayfish astacin and revealed that the cleaved human inhibitor blocks crayfish astacin and human meprin α and ß only slightly less potently than the intact variant. Therefore, the CTR of animal fetuin-B orthologs may have a function in maintaining a particular relative orientation of CY1 and CY2 that nonetheless is dispensable for peptidase inhibition.

Recombinant production, purification, crystallization, and structure analysis of human transforming growth factor ß2 in a new conformation.

Transforming growth factor ß is a disulfide-linked dimeric cytokine that occurs in three highly related isoforms (TGFß1-TGFß3) engaged in signaling functions through binding of cognate TGFß receptors. To regulate this pathway, the cytokines are biosynthesized as inactive pro-TGFßs with an N-terminal latency-associated protein preceding the mature moieties. Due to their pleiotropic implications in physiology and pathology, TGFßs are privileged objects of in vitro studies. However, such studies have long been limited by the lack of efficient human recombinant expression systems of native, glycosylated, and homogenous proteins. Here, we developed pro-TGFß2 production systems based on human Expi293F cells, which yielded >2 mg of pure histidine- or Strep-tagged protein per liter of cell culture. We assayed this material biophysically and in crystallization assays and obtained a different crystal form of mature TGFß2, which adopted a conformation deviating from previous structures, with a distinct dimeric conformation that would require significant rearrangement for binding of TGFß receptors. This new conformation may be reversibly adopted by a certain fraction of the mature TGß2 population and represent a hitherto undescribed additional level of activity regulation of the mature growth factor once the latency-associated protein has been separated.