Proteolytic cleavage of the TGFß co-receptor CD109 changes its conformation, resulting in protease inhibition via activation of its thiol ester, and dissociation from the cell membrane.

The glycosylphosphatidylinositol (GPI)-anchored protein cluster of differentiation 109 (CD109) is expressed on many human cell types and modulates the transforming growth factor ß (TGF-ß) signaling network. CD109 belongs to the alpha-macroglobulin family of proteins, known for their protease-triggered conformational changes. However, the effect of proteolysis on CD109 and its conformation are unknown. Here, we investigated the interactions of CD109 with proteases. We found that a diverse selection of proteases cleaved peptide bonds within the predicted bait region of CD109, inducing a conformational change that activated the thiol ester of CD109. We show CD109 was able to conjugate proteases with this thiol ester and decrease their activity toward protein substrates, demonstrating that CD109 is a protease inhibitor. We additionally found that CD109 has a unique mechanism whereby its GPI-anchored macroglobulin 8 (MG8) domain dissociates during its conformational change, allowing proteases to release CD109 from the cell surface by a precise mechanism and not unspecific shedding. We conclude that proteolysis of the CD109 bait region affects both its structure and location, and that interactions between CD109 and proteases may be important to understanding its functions, for example, as a TGF-ß co-receptor.

Characterization of a novel cold-adapted intracellular serine protease from the extremophile Planococcus halocryophilus Or1.

The enzymes of microorganisms that live in cold environments must be able to function at ambient temperatures. Cold-adapted enzymes generally have less ordered structures that convey a higher catalytic rate, but at the cost of lower thermodynamic stability. In this study, we characterized P355, a novel intracellular subtilisin protease (ISP) derived from the genome of Planococcus halocryophilus Or1, which is a bacterium metabolically active down to -25°C. P355's stability and activity at varying pH values, temperatures, and salt concentrations, as well as its temperature-dependent kinetics, were determined and compared to an uncharacterized thermophilic ISP (T0099) from Parageobacillus thermoglucosidasius, a previously characterized ISP (T0034) from Planococcus sp. AW02J18, and Subtilisin Carlsberg (SC). The results showed that P355 was the most heat-labile of these enzymes, closely followed by T0034. P355 and T0034 exhibited catalytic constants (k cat ) that were much higher than those of T0099 and SC. Thus, both P355 and T0034 demonstrate the characteristics of the stability-activity trade-off that has been widely observed in cold-adapted proteases.

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.

A top-down approach to uncover the hidden ligandome of low-density lipoprotein receptor-related protein 1 in cartilage.

The low-density lipoprotein receptor-related protein 1 (LRP1) is a cell-surface receptor ubiquitously expressed in various tissues. It plays tissue-specific roles by mediating endocytosis of a diverse range of extracellular molecules. Dysregulation of LRP1 is involved in multiple conditions including osteoarthritis (OA) but little information is available about the specific profile of direct binding partners of LRP1 (ligandome) for each tissue, which would lead to a better understanding of its role in disease states. Here, we investigated adult articular cartilage where impaired LRP1-mediated endocytosis leads to tissue destruction. We used a top-down approach involving proteomic analysis of the LRP1 interactome in human chondrocytes, direct binding assays using purified LRP1 and ligand candidates, and validation in LRP1-deficient fibroblasts and human chondrocytes, as well as a novel Lrp1 conditional knockout (KO) mouse model. We found that inhibition of LRP1 and ligand interaction results in cell death, alteration of the entire secretome and transcriptional modulations in human chondrocytes. We identified a chondrocyte-specific LRP1 ligandome consisting of more than 50 novel ligand candidates. Surprisingly, 23 previously reported LRP1 ligands were not regulated by LRP1-mediated endocytosis in human chondrocytes. We confirmed direct LRP1 binding of HGFAC, HMGB1, HMGB2, CEMIP, SLIT2, ADAMTS1, TSG6, IGFBP7, SPARC and LIF, correlation between their affinity for LRP1 and the rate of endocytosis, and some of their intracellular localization. Moreover, a conditional LRP1 KO mouse model demonstrated a critical role of LRP1 in regulating the high-affinity ligands in cartilage in vivo. This systematic approach revealed the specificity and the extent of the chondrocyte LRP1 ligandome and identified potential novel therapeutic targets for OA.

Cryo-EM structures of human A2ML1 elucidate the protease-inhibitory mechanism of the A2M family.

A2ML1 is a monomeric protease inhibitor belonging to the A2M superfamily of protease inhibitors and complement factors. Here, we investigate the protease-inhibitory mechanism of human A2ML1 and determine the structures of its native and protease-cleaved conformations. The functional inhibitory unit of A2ML1 is a monomer that depends on covalent binding of the protease (mediated by A2ML1's thioester) to achieve inhibition. In contrast to the A2M tetramer which traps proteases in two internal chambers formed by four subunits, in protease-cleaved monomeric A2ML1 disordered regions surround the trapped protease and may prevent substrate access. In native A2ML1, the bait region is threaded through a hydrophobic channel, suggesting that disruption of this arrangement by bait region cleavage triggers the extensive conformational changes that result in protease inhibition. Structural comparisons with complement C3/C4 suggest that the A2M superfamily of proteins share this mechanism for the triggering of conformational change occurring upon proteolytic activation.

The low-density lipoprotein receptor-related protein 1 (LRP1) interactome in the human cornea.

The human cornea is responsible for approximately 70% of the eye's optical power and, together with the lens, constitutes the only transparent tissue in the human body. Low-density lipoprotein receptor-related protein 1 (LRP1), a large, multitalented endocytic receptor, is expressed throughout the human cornea, yet its role in the cornea remains unknown. More than 30 years ago, LRP1 was purified by exploiting its affinity for the activated form of the protease inhibitor alpha-2-macroblulin (A2M), and the original purification protocol is generally referred to in studies involving full-length LRP1. Here, we provide a novel and simplified LRP1 purification protocol based on LRP1's affinity for receptor-related protein (RAP) that produces significantly higher yields of authentic LRP1. Purified LRP1 was used to map its unknown interactome in the human cornea. Corneal proteins extracted under physiologically relevant conditions were subjected to LRP1 affinity pull-down, and LRP1 ligand candidates were identified by LC-MS/MS. A total of 28 LRP1 ligand candidates were found, including 22 novel ligands. The LRP1 corneal interactome suggests a novel role for LRP1 as a regulator of the corneal immune response, structure, and ultimately corneal transparency.

Latency, thermal stability, and identification of an inhibitory compound of mirolysin, a secretory protease of the human periodontopathogen Tannerella forsythia.

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.

ITIH4 acts as a protease inhibitor by a novel inhibitory mechanism.

Inter-α-inhibitor heavy chain 4 (ITIH4) is a poorly characterized plasma protein that is proteolytically processed in multiple pathological conditions. However, no biological function of ITIH4 has been identified. Here, we show that ITIH4 is cleaved by several human proteases within a protease-susceptible region, enabling ITIH4 to function as a protease inhibitor. This is exemplified by its inhibition of mannan-binding lectin-associated serine protease-1 (MASP-1), MASP-2, and plasma kallikrein, which are key proteases for intravascular host defense. Mechanistically, ITIH4 acts as bait that, upon cleavage, forms a noncovalent, inhibitory complex with the executing protease that depends on the ITIH4 von Willebrand factor A domain. ITIH4 inhibits the MASPs by sterically preventing larger protein substrates from accessing their active sites, which remain accessible and fully functional toward small substrates. Thus, we demonstrate that ITIH4 functions as a protease inhibitor by a previously undescribed inhibitory mechanism.

α2-Macroglobulin-like protein 1 can conjugate and inhibit proteases through their hydroxyl groups, because of an enhanced reactivity of its thiol ester.

Proteins in the α-macroglobulin (αM) superfamily use thiol esters to form covalent conjugation products upon their proteolytic activation. αM protease inhibitors use theirs to conjugate proteases and preferentially react with primary amines (e.g. on lysine side chains), whereas those of αM complement components C3 and C4B have an increased hydroxyl reactivity that is conveyed by a conserved histidine residue and allows conjugation to cell surface glycans. Human α2-macroglobulin-like protein 1 (A2ML1) is a monomeric protease inhibitor but has the hydroxyl reactivity-conveying histidine residue. Here, we have investigated the role of hydroxyl reactivity in a protease inhibitor by comparing recombinant WT A2ML1 and the A2ML1 H1084N mutant in which this histidine is removed. Both of A2ML1s' thiol esters were reactive toward the amine substrate glycine, but only WT A2ML1 reacted with the hydroxyl substrate glycerol, demonstrating that His-1084 increases the hydroxyl reactivity of A2ML1's thiol ester. Although both A2ML1s conjugated and inhibited thermolysin, His-1084 was required for the conjugation and inhibition of acetylated thermolysin, which lacks primary amines. Using MS, we identified an ester bond formed between a thermolysin serine residue and the A2ML1 thiol ester. These results demonstrate that a histidine-enhanced hydroxyl reactivity can contribute to protease inhibition by an αM protein. His-1084 did not improve A2ML1's protease inhibition at pH 5, indicating that A2ML1's hydroxyl reactivity is not an adaption to its acidic epidermal environment.

Kallikrein-Related Peptidase 14 Activates Zymogens of Membrane Type Matrix Metalloproteinases (MT-MMPs)-A CleavEx Based Analysis.

Kallikrein-related peptidases (KLKs) and matrix metalloproteinases (MMPs) are secretory proteinases known to proteolytically process components of the extracellular matrix, modulating the pericellular environment in physiology and in pathologies. The interconnection between these families remains elusive. To assess the cross-activation of these families, we developed a peptide, fusion protein-based exposition system (Cleavage of exposed amino acid sequences, CleavEx) aiming at investigating the potential of KLK14 to recognize and hydrolyze proMMP sequences. Initial assessment identified ten MMP activation domain sequences which were validated by Edman degradation. The analysis revealed that membrane-type MMPs (MT-MMPs) are targeted by KLK14 for activation. Correspondingly, proMMP14-17 were investigated in vitro and found to be effectively processed by KLK14. Again, the expected neo-N-termini of the activated MT-MMPs was confirmed by Edman degradation. The effectiveness of proMMP activation was analyzed by gelatin zymography, confirming the release of fully active, mature MT-MMPs upon KLK14 treatment. Lastly, MMP14 was shown to be processed on the cell surface by KLK14 using murine fibroblasts overexpressing human MMP14. Herein, we propose KLK14-mediated selective activation of cell-membrane located MT-MMPs as an additional layer of their regulation. As both, KLKs and MT-MMPs, are implicated in cancer, their cross-activation may constitute an important factor in tumor progression and metastasis.

Plasmin inhibition by bacterial serpin: Implications in gum disease.

Tannerella forsythia is a periodontopathogen that expresses miropin, a protease inhibitor in the serpin superfamily. In this study, we show that miropin is also a specific and efficient inhibitor of plasmin; thus, it represents the first proteinaceous plasmin inhibitor of prokaryotic origin described to date. Miropin inhibits plasmin through the formation of a stable covalent complex triggered by cleavage of the Lys368-Thr369 (P2-P1) reactive site bond with a stoichiometry of inhibition of 3.8 and an association rate constant (kass) of 3.3 × 105 M-1s-1. The inhibition of the fibrinolytic activity of plasmin was nearly as effective as that exerted by α2-antiplasmin. Miropin also acted in vivo by reducing blood loss in a mice tail bleeding assay. Importantly, intact T. forsythia cells or outer membrane vesicles, both of which carry surface-associated miropin, strongly inhibited plasmin. In intact bacterial cells, the antiplasmin activity of miropin protects envelope proteins from plasmin-mediated degradation. In summary, in the environment of periodontal pockets, which are bathed in gingival crevicular fluid consisting of 70% of blood plasma, an abundance of T. forsythia in the bacterial biofilm can cause local inhibition of fibrinolysis, which could have possible deleterious effects on the tooth-supporting structures of the periodontium.

Matrix-degrading protease ADAMTS-5 cleaves inter-α-inhibitor and releases active heavy chain 2 in synovial fluids from arthritic patients.

Destruction of the cartilage matrix in joints is an important feature of arthritis. Proteolytic degradation of cartilage glycoproteins can contribute to the loss of matrix integrity. Human inter-α-inhibitor (IαI), which stabilizes the extracellular matrix, is composed of the light-chain serine proteinase inhibitor bikunin and two homologous heavy chains (HC1 and HC2) covalently linked through chondroitin 4-sulfate. Inflammation promotes the transfer of HCs from chondroitin 4-sulfate to hyaluronan by tumor necrosis factor-stimulated gene-6 protein (TSG-6). This reaction generates a covalent complex between the heavy chains and hyaluronan that can promote leukocyte invasion. This study demonstrates that both IαI and the HC-hyaluronan complex are substrates for the extracellular matrix proteases ADAMTS-5 and matrix metalloprotease (MMP) -3, -7, and -13. The major cleavage sites for all four proteases are found in the C terminus of HC2. ADAMTS-5 and MMP-7 displayed the highest activity toward HC2. ADAMTS-5 degradation products were identified in mass spectrometric analysis of 29 of 33 arthropathic patients, indicating that ADAMTS-5 cleavage occurs in synovial fluid in arthritis. After cleavage, free HC2, together with TSG-6, is able to catalyze the transfer of heavy chains to hyaluronan. The release of extracellular matrix bound HC2 is likely to increase the mobility of the HC2/TSG-6 catalytic unit and consequently increase the rate of the HC transfer reaction. Ultimately, ADAMTS-5 cleavage of HC2 could alter the physiological and mechanical properties of the extracellular matrix and contribute to the progression of arthritis.

Proteolytic processing and activation of gingipain zymogens secreted by T9SS of Porphyromonas gingivalis.

Porphyromonas gingivalis uses a type IX secretion system (T9SS) to deliver more than 30 proteins to the bacterial surface using a conserved C-terminal domain (CTD) as an outer membrane translocation signal. On the surface, the CTD is cleaved and an anionic lipopolysaccharide (A-PLS) is attached by PorU sortase. Among T9SS cargo proteins are cysteine proteases, gingipains, which are secreted as inactive zymogens requiring removal of an inhibiting N-terminal prodomain (PD) for activation. Here, we have shown that the gingipain proRgpB isolated from the periplasm of a T9SS-deficient P. gingivalis strain was stable and did not undergo autocatalytic activation. Addition of purified, active RgpA or RgpB, but not Lys-specific Kgp, efficiently cleaved the PD of proRgpB but catalytic activity remained inhibited because of inhibition of the catalytic domain in trans by the PD. In contrast, active RgpB was generated from the zymogen, although at a slow rate, by gingipain-null P. gingivalis lysate or intact bacterial cell suspension. This activation was dependent on the presence of the PorU sortase. Interestingly, maturation of proRgpB with the catalytic cysteine residues mutated to Ala expressed in the ΔRgpA mutant strain was indistinguishable from that in the parental strain. Cumulatively, this suggests that PorU not only has sortase activity but is also engaged in activation of gingipain zymogens on the bacterial cell surface.

Structural and Functional Implications of Human Transforming Growth Factor ß-Induced Protein, TGFBIp, in Corneal Dystrophies.

A major cause of visual impairment, corneal dystrophies result from accumulation of protein deposits in the cornea. One of the proteins involved is transforming growth factor ß-induced protein (TGFBIp), an extracellular matrix component that interacts with integrins but also produces corneal deposits when mutated. Human TGFBIp is a multi-domain 683-residue protein, which contains one CROPT domain and four FAS1 domains. Its structure spans ∼120 Å and reveals that vicinal domains FAS1-1/FAS1-2 and FAS1-3/FAS1-4 tightly interact in an equivalent manner. The FAS1 domains are sandwiches of two orthogonal four-stranded ß sheets decorated with two three-helix insertions. The N-terminal FAS1 dimer forms a compact moiety with the structurally novel CROPT domain, which is a five-stranded all-ß cysteine-knot solely found in TGFBIp and periostin. The overall TGFBIp architecture discloses regions for integrin binding and that most dystrophic mutations cluster at both molecule ends, within domains FAS1-1 and FAS1-4.

Analysis of Factor D Isoforms in Malpuech-Michels-Mingarelli-Carnevale Patients Highlights the Role of MASP-3 as a Maturase in the Alternative Pathway of Complement.

Factor D (FD), which is also known as adipsin, is regarded as the first-acting protease of the alternative pathway (AP) of complement. It has been suggested that FD is secreted as a mature enzyme that does not require subsequent activation. This view was challenged when it was shown that mice lacking mannose-binding lectin (MBL)-associated serine protease-1 (MASP-1) and MASP-3 contain zymogenic FD (pro-FD), and it is becoming evident that MASP-3 is implicated in pro-FD maturation. However, the necessity of MASP-3 for pro-FD cleavage has been questioned, because AP activity is still observed in sera from MASP-1/3-deficient Malpuech-Michels-Mingarelli-Carnevale (3MC) patients. The identification of a novel 3MC patient carrying a previously unidentified MASP-3 G665S mutation prompted us to develop an analytical isoelectric focusing technique that resolves endogenous FD variants in complex samples. This enabled us to show that although 3MC patients predominantly contain pro-FD, they also contain detectable levels of mature FD. Moreover, using isoelectric focusing analysis, we show that both pro-FD and FD are present in the circulation of healthy donors. We characterized the naturally occurring 3MC-associated MASP-3 mutants and found that they all yielded enzymatically inactive proteins. Using MASP-3-depleted human serum, serum from 3MC patients, and Masp1/3-/- mice, we found that lack of enzymatically active MASP-3, or complete MASP-3 deficiency, compromises the conversion of pro-FD to FD. In summary, our observations emphasize that MASP-3 acts as an important maturase in the AP of complement, while also highlighting that there exists MASP-3-independent pro-FD maturation in 3MC patients.

Mirolysin, a LysargiNase from Tannerella forsythia, proteolytically inactivates the human cathelicidin, LL-37.

Tannerella forsythia is a periodontal pathogen expressing six secretory proteolytic enzymes with a unique multidomain structure referred to as KLIKK proteases. Two of these proteases, karilysin and mirolysin, were previously shown to protect the bacterium against complement-mediated bactericidal activity. The latter metalloprotease, however, was not characterized at the protein level. Therefore, we purified recombinant mirolysin and subjected it to detailed biochemical characterization. Mirolysin was obtained as a 66 kDa zymogen, which autoproteolytically processed itself into a 31 kDa active form via truncations at both the N- and C-termini. Further autodegradation was prevented by calcium. Substrate specificity was determined by the S1' subsite of the substrate-binding pocket, which shows strong preference for Arg and Lys at the carbonyl side of a scissile peptide bond (P1' residue). The protease cleaved an array of host proteins, including human fibronectin, fibrinogen, complement proteins C3, C4, and C5, and the antimicrobial peptide, LL-37. Degradation of LL-37 abolished not only the bactericidal activity of the peptide, but also its ability to bind lipopolysaccharide (LPS), thus quenching the endotoxin proinflammatory activity. Taken together, these results indicate that, through cleavage of LL-37 and complement proteins, mirolysin might be involved in evasion of the host immune response.

Structural and functional probing of PorZ, an essential bacterial surface component of the type-IX secretion system of human oral-microbiomic Porphyromonas gingivalis.

Porphyromonas gingivalis is a member of the human oral microbiome abundant in dysbiosis and implicated in the pathogenesis of periodontal (gum) disease. It employs a newly described type-IX secretion system (T9SS) for secretion of virulence factors. Cargo proteins destined for secretion through T9SS carry a recognition signal in the conserved C-terminal domain (CTD), which is removed by sortase PorU during translocation. Here, we identified a novel component of T9SS, PorZ, which is essential for surface exposure of PorU and posttranslational modification of T9SS cargo proteins. These include maturation of enzyme precursors, CTD removal and attachment of anionic lipopolysaccharide for anchorage in the outer membrane. The crystal structure of PorZ revealed two ß-propeller domains and a C-terminal ß-sandwich domain, which conforms to the canonical CTD architecture. We further documented that PorZ is itself transported to the cell surface via T9SS as a full-length protein with its CTD intact, independently of the presence or activity of PorU. Taken together, our results shed light on the architecture and possible function of a novel component of the T9SS. Knowledge of how T9SS operates will contribute to our understanding of protein secretion as part of host-microbiome interactions by dysbiotic members of the human oral cavity.

Disulfide Bond Pattern of Transforming Growth Factor ß-Induced Protein.

Transforming growth factor ß-induced protein (TGFBIp) is an extracellular matrix protein composed of an NH2-terminal cysteine-rich domain (CRD) annotated as an emilin (EMI) domain and four fasciclin-1 (FAS1-1-FAS1-4) domains. Mutations in the gene cause corneal dystrophies, a group of debilitating protein misfolding diseases that lead to severe visual impairment. Previous studies have shown that TGFBIp in the cornea is cross-linked to type XII collagen through a reducible bond. TGFBIp contains 11 cysteine residues and is thus able to form five intramolecule disulfide bonds, leaving a single cysteine residue available for the collagen cross-link. The structures of TGFBIp and its homologues are unknown. We here present the disulfide bridge pattern of TGFBIp, which was determined by generating specific peptides. These were separated by ion exchange followed by reversed-phase high-performance liquid chromatography and analyzed by mass spectrometry and Edman degradation. The NH2-terminal CRD contains six cysteine residues, and one of these (Cys65) was identified as the candidate for the reducible cross-link between TGFBIp and type XII collagen. In addition, the CRD contained two intradomain disulfide bridges (Cys49-Cys85 and Cys84-Cys97) and one interdomain disulfide bridge to FAS1-2 (Cys74-Cys339). Significantly, this arrangement violates the predicted disulfide bridge pattern of an EMI domain. The cysteine residues in FAS1-3 (Cys473 and Cys478) were shown to form an intradomain disulfide bridge. Finally, an interdomain disulfide bridge between FAS1-1 and FAS1-2 (Cys214-Cys317) was identified. The interdomain disulfide bonds indicate that the NH2 terminus of TGFBIp (CRD, FAS1-1, and FAS1-2) adopts a compact globular fold, leaving FAS1-3 and FAS1-4 exposed.

The outer-membrane export signal of Porphyromonas gingivalis type IX secretion system (T9SS) is a conserved C-terminal ß-sandwich domain.

In the recently characterized Type IX Secretion System (T9SS), the conserved C-terminal domain (CTD) in secreted proteins functions as an outer membrane translocation signal for export of virulence factors to the cell surface in the Gram-negative Bacteroidetes phylum. In the periodontal pathogen Porphyromonas gingivalis, the CTD is cleaved off by PorU sortase in a sequence-independent manner, and anionic lipopolysaccharide (A-LPS) is attached to many translocated proteins, thus anchoring them to the bacterial surface. Here, we solved the atomic structure of the CTD of gingipain B (RgpB) from P. gingivalis, alone and together with a preceding immunoglobulin-superfamily domain (IgSF). The CTD was found to possess a typical Ig-like fold encompassing seven antiparallel ß-strands organized in two ß-sheets, packed into a ß-sandwich structure that can spontaneously dimerise through C-terminal strand swapping. Small angle X-ray scattering (SAXS) revealed no fixed orientation of the CTD with respect to the IgSF. By introducing insertion or substitution of residues within the inter-domain linker in the native protein, we were able to show that despite the region being unstructured, it nevertheless is resistant to general proteolysis. These data suggest structural motifs located in the two adjacent Ig-like domains dictate the processing of CTDs by the T9SS secretion pathway.