Dissecting Cytophagalysin: Structural and Biochemical Studies of a Bacterial Pappalysin-Family Metallopeptidase

Citation: Estevan-Morió, E.; Ramírez-Larrota, J.S.; Bushi, E.; Eckhard, U. Dissecting Cytophagalysin: Structural and Biochemical Studies of a Bacterial Pappalysin-Family Metallopeptidase. Biomolecules 2024,14, 1604. https:// doi.org/10.3390/biom14121604 Academic Editor: Pierre Lafite Received: 24 November 2024 Revised: 7 December 2024 Accepted: 12 December 2024 Published: 16 December 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Article Dissecting Cytophagalysin: Structural and Biochemical Studies of a Bacterial Pappalysin-Family Metallopeptidase Eva Estevan-Morió1,2 , Juan Sebastián Ramírez-Larrota 1,2 , Enkela Bushi 1and Ulrich Eckhard 1,∗ 1Synthetic Structural Biology Group, Molecular Biology Institute of Barcelona (IBMB), Spanish National Research Council (CSIC), 08028 Barcelona, Spain 2Doctorate in Biotechnology, Faculty of Pharmacy and Food Sciences, University of Barcelona, 08028 Barcelona, Spain *Correspondence: ulrich.eckhar[email protected] Abstract: Cytophaga is a genus of Gram-negative bacteria occurring in soil and the gut microbiome. It is closely related to pathogenic Flavobacterium spp. that cause severe diseases in fish. Cytophaga strain L43-1 secretes cytophagalysin (CPL1), a 137 kDa peptidase with reported collagenolytic and gelatinolytic activity. We performed highly-confident structure prediction calculations for CPL1, which identified 11 segments and domains, including a signal peptide for secretion, a prosegment (PS) for latency, a metallopeptidase (MP)-like catalytic domain (CD), and eight immunoglobulin (Ig)-like domains (D3–D10). In addition, two short linkers were found at the D8–D9 and D9–D10 junctions, and the structure would be crosslinked by four disulfide bonds. The CPL1 CD was found closest to ulilysin from Methanosarcina acetivorans, which assigns CPL1 to the lower-pappalysin family within the metzincin clan of MPs. Based on the structure predictions, we aimed to produce constructs spanning the full-length enzyme, as well as PS+CD, PS+CD+D3, and PS+CD+D3+D4. However, we were successful only with the latter three constructs. We could activate recombinant CPL1 by PS removal employing trypsin, and found that both zymogen and mature CPL1 were active in gelatin zymography and against a fluorogenic gelatin variant. This activity was ablated in a mutant, in which the catalytic glutamate described for lower pappalyins and other metzincins was replaced by alanine, and by a broad-spectrum metal chelator. Overall, these results proved that our recombinant CPL1 is a functional active MP, thus supporting the conclusions derived from the structure predictions. Keywords: metallopeptidase; pappalysin family; recombinant protein expression; protease activation; functional characterization 1. Introduction Zinc-dependent metallopeptidases (MP) are ubiquitous peptidoand proteolytic catalysers engaged in a plethora of biochemical pathways [ 1 ], and they are subdivided into numerous families and clans. These include the aspzincin [ 2 ], gluzincin [ 3 ], and metzincin [ 4 ] clans, which together with S2P-zincins [ 5 , 6 ], FtsH-like AAA MPs [ 7 ], and iron-dependent peptide deformylase [ 8 ] form the zincin tribe of MPs [ 6 , 9 ]. Characteristic of zincins is a short motif (H–E–x–x–H; amino-acid one-letter-code, x for any residue), which includes two zinc-binding histidines and a catalytic glutamate. The latter acts first as a general base and then as a general acid during the catalytic mechanism, which pivots around the nucleophilic attack of a zinc-bound catalytic solvent molecule onto the scissile bond of a peptidic substrate bound to the active-site cleft [ 1 , 6 , 9 ]. Glutamate replacement with alanine hampers catalysis but maintains the active-site geometry [ 9 – 12 ], so such mutants are often used to create inactive catalytic domains or zymogens for structural and binding studies [ 9 ]. The metzincin clan, in turn, is hallmarked by a C-terminal extension of the motif (H–E–x–x–H–x–x–G/N–x–x–H/D), which includes a further glycine or asparagine essential for a conserved turn of the polypeptide chain that leads to a third zinc ligand three positions downstream in sequence, either a histidine or an aspartate [ 6 , 9 , 13 , 14 ]. At the Biomolecules 2024,14, 1604. https://doi.org/10.3390/biom14121604 https://www.mdpi.com/journal/biomolecules Biomolecules 2024,14, 1604 2 of 20 structural level, metzincins share a compact ellipsoidal catalytic domain (CD) of ~130–410 residues divided by the active-site cleft into an upper N-terminal subdomain (NSD) and a lower C-terminal subdomain (CSD) when pictured in the consensus standard orientation of MPs [ 15 ]. Metzincin NSDs share an “active-site helix”, which encompasses the first half of the extended zinc-binding motif, a “backing helix”, and a four/five-stranded β -sheet, whose lowermost β -strand forms the “upper rim” of the active-site cleft. CSDs share a “C-terminal helix” and a “Met-turn” centred on a conserved methionine residue that creates a hydrophobic base for the active-site zinc. Within metzincins, the pappalysin family [ 16 ] is named after its founding member, human pappalysin-1 or pregnancy-associated plasma protein-A, which is a ~180 kDa multidomain protein first reported from the bloodstream of pregnant women [ 17 , 18 ] whose cryo-electron microscopy structure has been recently elucidated [ 12 , 19 ]. Pappalysins are also grouped within family M43 in the MEROPS peptidase database (www.ebi.ac.uk/ merops; accessed on 20 November 2024) [ 20 ], and one subfamily features the “lower pappalysins” (LOPAPs) [ 21 ], previously referred to as “unicellular pappalysins” [ 22 , 23 ]. LOPAPs encompass similar sequences from archaea, bacteria, and, more recently, also unicellular and multicellular lower eukaryotes like algae and fungi [ 21 ]. LOPAPs share at least a short N-terminal prosegment (PS) for zymogenicity, which is a common feature of most MPs [ 9 ], and the zinc-dependent metzincin-type CD. Over the years, subfamily members have been described at the transcriptional, functional, and/or biochemical levels (see Table 1 in [ 21 ]), among which two have also been analysed for their crystal structures: mirolysin from the periodontopathogenic Gram-negative bacterium Tannerella forsythia and ulilysin from the environmental methanogenic archaeon Methanosarcina acetivorans [ 21 – 27 ]. The latter, also known as lysargiNase [ 21 , 28 ], is frequently used in bottom-up proteomics due to its trypsin-mirroring cleavage site specificity. Cytophaga is a bacterial genus that belongs to the Bacteroidetes phylum, formerly referred to as the CFB (Cytophaga,Bacteroides,Flavobacterium) phylum [ 29 ], which is dominant in gut microbial communities and inhabits the oral cavity, gastrointestinal tract, and urogenital tract of humans [ 30 ]. Cytophaga sp. strain L43-1, which is closely related to the Flavobacterium genus [ 31 ], secretes cytophagalysin (CPL1), a 1282-residue multi-modular LOPAP originally purified from culture supernatant and characterized [ 32 ]. Moreover, it was cloned and sequenced (see [ 33 , 34 ]) and is listed within the UniProt database (UP) [ 35 ] under entry Q46348. The purified enzyme was shown to cleave β -casein, both insoluble and acid-soluble collagens and gelatin, optimally at pH 7.5 and 30 ◦ C, but not small fluorogenic peptides that are cleaved by other bacterial collagenases [34]. Closely related to CPL1, an orthologue (UP A0A2H1E968) is encoded by Tenacibaculum maritimum NCIMB 2154 [ 36 ], a devastating pathogenic flavobacterium that causes tenacibaculosis in wild and farmed marine fish. Another pathogen, Flavobacterium psychrophylum, causes cold-water disease in Salmonidae and Osmeridae fish and encodes the 960-residue MPs WA-1 and WA-2, which are ~50% identical in sequence to CPL1 [ 31 ]. Other potential orthologues are encoded by the environmental bacteria Sediminitomix flava (UP A0A315ZEZ8) from marine sediment and Chitinophaga solisilvae (UP A0A3S1B1Z0) from forest soil. Importantly, any type of gelatinase or collagenase, including cytophagalysin, represents highly sought-after enzymes for biotechnology. Its ability to degrade collagen makes it valuable in medical tissue engineering through collagen remodelling. Collagenases play a crucial role in environmental bioremediation by degrading collagenand gelatin-based materials, thereby facilitating their utilization in the leather processing and food production industries. Here, we aimed to shed light on the structure and function of CPL1 and performed structure-prediction calculations. We analysed these results in the light of the accumulated knowledge on MPs in general and LOPAPs in particular. We further designed CPL1 constructs spanning distinct domains and achieved their recombinant overexpression and purification. Finally, we functionally assessed them in peptideand proteolytic assays in vitro, and performed mutants to validate their activity. Biomolecules 2024,14, 1604 3 of 20 2. Materials and Methods 2.1. Biocomputational Studies Sequence similarity searches and alignments were performed with the Blast algorithm [ 37 ] within UP and MultAlin [ 38 ], respectively, using standard parameters. The three-dimensional structure of full-length CPL1 was predicted with AlphaFold [ 39 ] using paired multiple sequence alignments, which enable the extraction of coevolutionary information and enhance the prediction accuracy [ 40 ]. The confidence of the five distinct predictions obtained was assessed by means of the predicted local-distance difference test (pLDDT [ 41 ]), which reliably estimates the accuracy of the C α local-distance difference test [ 39 ]. In this respect, pLDDT values >90% account for high accuracy of the overall prediction, and values >70% qualify as generally correct predictions of the backbone [ 42 ]. The models were visually inspected for chemical sense and coherence with Coot [ 43 ]. The top prediction (model_1) was subjected to manual model building to correct clashes and chemical inconsistencies, followed by geometrical regularisation with Coot. Thereafter, the Geometry_minimization routine of the Phenix suite [ 44 ] was applied, and the resulting final model, which can be downloaded as part of the Supplementary Materials, was validated with Molprobity [ 45 ] (Table S1). Inter-domain interfaces were analysed with Pisa [ 46 ] and structural superpositions were calculated using the Ssm routine [ 47 ] in Coot. Structural relatives present in the Protein Data Bank (PDB) were retrieved through Dali [ 48 ] and structural figures were prepared with Chimera [ 49 ] or an open-source build of PyMOL (Version 2.5 Schrödinger, LLC, New York, NY, USA) [50]. 2.2. Molecular Cloning of CPL1 Constructs The sequence of full-length CPL1 (Q 20 –K 1282 ) from Cytophaga sp. L43-1 31 was codonoptimized for expression in E. coli (Supplementary Figures S4 and S5), and a synthetic gene was purchased from GenScript Biotech for introduction into a modified pCri7a plasmid 66, hereafter pCri7a*, using the NdeI and BamHI restriction sites. This plasmid added an Nterminal Strep-tag with a recognition sequence for tobacco-etch virus peptidase (TEV) and a C-terminal His 8 -tag. Three C-terminally truncated variants of CPL1, designated CPL1_1-2 (Q 20 –N 328 ), CPL1_1-3 (Q 20 –V 444 ), and CPL1_1-4 (Q 20 –T 591 ), were subsequently cloned by inverse PCR [ 51 ] using 0.5 µ M of forward and reverse primers, 200 µ M of each of the four dNTPs, 0.02 U/ µ L Q5 High-Fidelity DNA Polymerase (New England Biolabs), but no High GC Enhancer. Constructs were produced as wild-type species and as mutants, in which the catalytic glutamate from the zinc-binding motif had been replaced with alanine (E 232 A). The two variants of CPL1_1-3 and CPL1_1-4 were also introduced into plasmid pCri7b [ 52 ], which only adds the C-terminal His 8 -tag, using the same restriction sites. Cloning and mutagenesis primers are listed under Supplementary Figure S6A,B, and the map of pCri7a* is shown under Supplementary Figure S6C. 2.3. Protein Expression and Purification Various chemically competent E. coli strains, including BL21(DE3), Origami 2(DE3), Lemo21(DE3), and Rosetta(DE3), were prepared inhouse following the Inoue method [ 53 ], transformed with the different protein-encoding plasmids, and plated on agar plates containing Bertani’s lysogeny broth [ 54 ] supplemented with kanamycin at 50 µ g/mL (hereafter medium). After overnight incubation, a single colony was picked for each strain and plasmid, and cultured in 5 mL medium at 37 ◦ C under gentle shaking (at 200 rpm). Cultures were then diluted 1:1000 in 500 mL fresh medium using four 2 L Erlenmeyer flasks, grown at 37 ◦ C to OD 600 ≈ 1.2, cooled for 15–30 min in a cold room, induced with 1 mM isopropylβ -D-1-thiogalactopyranoside (IPTG), and placed at 20 ◦ C under gentle shaking for overnight protein expression. Cells were subsequently harvested by centrifugation, resuspended in 30 mL buffer A (50 mM Tris · HCl pH 8.0, 300 mM sodium chloride, 10 mM imidazole, 10 mM calcium chloride, 50 µ M zinc chloride), and sonicated on ice for 1 min at 20% amplitude and 0.5 s intervals using a Branson 450 Digital Sonifier (Marshall Scientific, Hampton, NH, USA) with a 6 mm tapered microtip. The resulting Biomolecules 2024,14, 1604 4 of 20 solution was incubated on ice with magnesium chloride at 10 mM and 10 µ g/mL DNase I for 15 min, and then lysed using an E1061 continuous flow cell disruptor (Constant Systems, Daventry, UK) operated at 1.36 kbar. Lysates were incubated for 30 min on ice with 1 M urea before being centrifuged twice at 20,000 rpm using a JA-25.50 rotor in a Beckman Avanti J-25 centrifuge (~48,000 × g). The clarified supernatants were subjected to affinity chromatography employing HisPur Ni-NTA resin (Thermo Scientific, Waltham, MA, USA) and buffer A plus 20 mM or 250 mM imidazole for the wash or elution steps, respectively. Eluted samples were then concentrated using a Vivaspin-20 polyethersulfone centrifugal device with 10 kDa molecular-weight cutoff (Sartorius, Göttingen, Germany), and polished by SEC in Superdex 200 10/300 GL or Superose 6 Increase 10/300 GL columns (Cytiva, Marlborough, MA, USA), previously equilibrated with buffer B (50 mM Tris · HCl pH 8.0, 100 mM sodium chloride) and attached to an ÄKTApurifier 10 protein purification system (GE Healthcare, Milwaukee, WI, USA). Peak fractions were pooled, re-concentrated by centrifugation, and flash-frozen in liquid nitrogen as 50 µ L and 10 µ L aliquots of 10 mg/mL and 1 mg/mL concentration, respectively, and stored at − 80 ◦ C until usage. Protein concentrations were determined using a BioDrop Duo+ microvolume spectrophotometer (BioChrom, Cambridge, UK) based on the absorbance measured at λ = 280 nm and the respective theoretical extinction coefficient calculated by ProtParam [55,56]. Protein purity was assessed by SDS-PAGE analysis on custom-made 12% and 14% Tris/Glycine gels followed by Coomassie Brilliant Blue (Sigma-Aldrich, Saint Louis, MO, USA) staining. The BlueStar Plus Prestained protein marker (Nippon Genetics EUROPE, Düren, Germany) was used as molecular-mass reference. 2.4. Activation of CPL1 For activation, the wild-type CPL1 constructs, which encompass the predicted PS for latency (Q 20 –A 66 ; see Section 3.2), were incubated with bovine trypsin, treated with N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK) to prevent extraneous chymotryptic activity (Sigma-Aldrich, Saint Louis, MO, USA), at a weight ratio of 1000:1 for 10–15 min at room temperature, and activation through PS removal was monitored by SDS-PAGE. Similarly, bovine α -chymotrypsin, treated with N-tosyl-L-lysinyl chloromethyl ketone (TLCK) to ablate undesired trypsin activity (Sigma-Aldrich, Saint Louis, MO, USA), was likewise used to assess activation. In either case, reactions were stopped by adding the EDTA-free HALT inhibitor cocktail (Thermo Fisher Scientific, Waltham, MA, USA) at a final concentration of 2 × , which is equivalent to 2 mM of the broad-spectrum serine peptidase inhibitor 4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF). 2.5. Activity Assessment by Zymography Activated CPL1 variants were loaded onto 12% or 14% SDS-PAGE gels containing 0.1% gelatin from cold water fish skin (Sigma-Aldrich) and subjected to electrophoresis at 160 V for ~1 h on ice. Thereafter, gels were extensively rinsed with distilled water and washed three times for 30 min in buffer C (50 mM Tris · HCl pH 8.0, 150 mM sodium chloride, 2.5% Triton X-100) under gentle shaking. After two further washes with buffer D (50 mM Tris · HCl pH 8.0, 150 mM sodium chloride, 10 mM calcium chloride, 50 µ M zinc chloride, 0.02% Brij-35) for 20 min, gels were incubated in the same buffer overnight at room temperature, and stained with Coomassie Brilliant Blue. After a brief destaining step, proteolytic activity was revealed as light bands against over a dark blue background. All samples for zymography were prepared with non-reducing sample buffer without heating if not stated otherwise. 2.6. Fluorometric Gelatinolytic Activity and Inhibition Activity of CPL1 variants against gelatin in solution was determined using the substrate DQ Gelatin from pig skin (Thermo Fisher Scientific, Waltham, MA, USA), which consists of highly quenched fluorescein-labelled gelatin that yields bright green fluorescence upon proteolysis of the substrate. Substrate at 20 µ g/mL in 100 µ L buffer E (50 mM Biomolecules 2024,14, 1604 5 of 20 Tris · HCl pH 8, 150 mM sodium chloride, 10 mM calcium chloride, 50 µ M zinc chloride, 1 × Halt TM Protease Inhibitor Cocktail; Thermo Fisher Scientific, Waltham, MA, USA) was reacted with CPL1 at 37 ◦ C, and the fluorescence response was monitored in a BioTek Synergy H1 multimode microplate reader (Agilent Technologies, Santa Clara, CA, USA) with λexc = 480 nm and λem = 520 nm. Activity inhibition was assessed by preincubating the CPL1 variants with 20 mM EDTA for 30 min and proceeding as mentioned. 2.7. Collagen Degradation Assay Activity of CPL1 variants against the VitroCol type-I human atelocollagen (Advanced Biomatrix, Carlsbad, CA, USA) and against rat-tail type-I collagen (Corning, Corning, NY, USA) was tested in 40 µ L reaction volumes by adding 2 µ g of enzyme to 10 µ g of collagen in buffer E. After 16 h of incubation at 20 ◦ C to ensure native collagen conditions, cleavage was assessed by 12% SDS-PAGE gels. Collagenase type IA from Clostridium histolyticum, bovine TPCK-treated trypsin, and bovine TLCK-treated α -chymotrypsin (all from Sigma-Aldrich, St. Louis, MO, USA) were used as controls. 2.8. Protein Crystallization Assays Crystallization assays were performed by the sitting-drop vapor diffusion method. Reservoir solutions were prepared by a Tecan robot and 100 nL crystallization drops were dispensed on 96-well 2-drop Swissci PS MRC plates (Molecular Dimensions, Sheffield, UK) by a Phoenix nanodrop robot (Art Robbins Instruments, Sunnyvale, CA, USA) or a Cartesian Microsys 4000 XL robot (Genomic Solutions, Ann Arbor, MI, USA) at the joint IBMB/IRB Automated Crystallography Platform (https://ibmb.csic.es/en/platforms/ automated-crystallographic-platform; accessed on 20 November 2024). Plates were stored in steady-temperature crystal farms (Bruker, Billerica, MA, USA) at 4 ◦ C or 20 ◦ C and regularly inspected. 3. Results and Discussion 3.1. Computational Prediction of the CPL1 Structure Similarity searches identified mirolysin (UP A0A0F7IPS1 and G8ULV1, wrongly entitled “karilysin”) and ulilysin (UP Q8TL28) as the closest sequences of CPL1 among structurally analysed proteins (p= 1.8 × 10 −23 , 269 common residues, 30% sequence identity; and p= 1.5 × 10 −20 , 237 common residues, 31% sequence identity, respectively). The aligned sequence stretches encompassed in both cases the respective PSs and CDs, which supports the ascription of CPL1 CD to the LOPAPs (Figure 1A). Next, we performed computational structure predictions for residues 1–1282 with the AlphaFold program [ 39 ], which were highly confident according to the predicted localdistance difference test (pLDDT) and sequence coverage (Figure 1C,D). The highest-ranking model of five predictions was subjected to manual rebuilding and geometric minimization. Supplementary Table S1 depicts the statistics of the validation of the resulting working model, which can be downloaded as part of the Supplementary Information. This model proposes the presence of 11 segments and domains plus two linkers, which include a 19-residue signal peptide for secretion (SP) followed by a 47-residue PS (Q 20 –A 66 ; sequence numbering of CPL1 in superscript, see UP Q46348) (Figure 1B) that is absent from the enzyme purified from culture supernatant [ 32 – 34 ]. Next, the metal-dependent CD (E67–S329) would contain two intradomain disulfides (C 247 –C 273 and C 267 –C 292 ) plus a third one (C 140 – C 434 ) that links this domain to downstream domain D3 (T 330 –P 446 ). The latter precedes seven further domains (D4, Y 447 –M 593 ; D5, T 594 –S 713 ; D6, P 714 –G 864 ; D7, I 865 –A 959 ; D8, S 960 –G 1099 ; D9, T 1107 –P 1194 ; and D10, K 1207 –K 1282 ), with two short linkers inserted at the D8–D9 and D9–D10 junctions (Figure 1B). Biomolecules 2024,14, 1604 6 of 20 Biomolecules 2024, 14, x FOR PEER REVIEW 6 of 20 S960–G1099; D9, T1107–P1194; and D10, K1207–K1282), with two short linkers inserted at the D8– D9 and D9–D10 junctions (Figure 1B). With respect to the relative orientations of the domains, only the first five domains consistently appeared in a comparable arrangement in the five predictions (see Figure 1E). This is consistent with the fact that relative orientations of domains cannot usually be predicted accurately [57]. Indeed, analysis of the predicted aligned error, which accounts for the residue–residue alignment confidence and, thus, if domains are reliably positioned [58], suggests that PS–CD, CD–D3, as well as D3–D4 and D4–D5 might be correctly placed relative to one another in the predictions (Figure 1F). Visual inspection and computational quantification of the inter-domain interfaces with Pisa [46] suggested that the relative arrangement of PS–CD and CD–D3 might actually be significant. It features interfaces spanning 1329 Å2 and 518 Å2, respectively, which are in the range of values reported for experimental protein–protein complexes (955 ± 380 Å2) [59], and the calculated ΔiG values are - 12.2 kcal/mol and -8.9 kcal/mol, respectively. In contrast, the D3–D4 and D4–D5 interfaces feature only few contacts and may rather represent hinge points for structure rearrangement. Future studies may include multi-state small-angle X-ray scattering (SAXS) profile analysis of near-to-full-length protein preparations to experimentally capture the structural interplay of accessory domains. Figure 1. Biocomputational studies. (A) Sequence alignment of the prosegments (PSs) (green background) and catalytic domains (CDs) of CPL1 (UP Q46348), mirolysin (UP G8ULV1), and ulilysin (UP Q8TL28). Identical or equivalent residues are in red, and those shared by two sequences are in blue. The PS cysteine engaged in zinc-binding in the zymogen—putatively in CPL1—is framed. The extended zinc-binding motif, the residues shaping the common calcium site, and the Met-turn motif are shown over light-blue, orange, and cyan background, respectively. (B) Domain distribution along the chemical sequence predicted by AlphaFold, which foresees a signal peptide for secretion (SP), the PS, the CD, and immunoglobulin-like domains D3 through D10. Each domain is labelled, the respective limiting residues are indicated, and the average predicted local-distance difference test (pLDDT) is shown in parenthesis. In all cases, these values are close to or exceed the high-accuracy cut-off of ~90% [ 42 ], and are thus classed as high confidence. The only exception is the PS, whose prediction evinces an average pLDDT that is slightly lower, but still highly reliable for the main chain. Two short linkers (LNKs) would be intercalated between D8 and D9, and between D9 and D10. Predicted disulfide bonds are shown in orange. The cysteine putatively engaged in latency in the zymogen (C 24 ) and the extended zinc-binding motif (H 231 –H 241 ), as well as the Met-turn methionine (M 284 ) and the maturation cleavage point (A 66 –E 67 ; scissors) are further pinpointed. (C) pLDDT for each residue of the prediction (positions 1–1282) for each of the five distinct models obtained. (D) Sequence coverage for each residue of the prediction (positions 1–1282) vs. number of sequences. Biomolecules 2024,14, 1604 7 of 20 (E) Superposition of the five predicted models without further relaxation/minimization with each domain/segment in the colour of (B). Only PS, CD, D3, D4, and, roughly, D5 appear with similar relative orientations in all models. (F) Analysis of the predicted aligned error, which estimates if domains are correctly positioned relative to one another, for each residue of the prediction (positions 1–1282; model_1). Each segment/domain of (B) gives rise to a marine blue square along the diagonal. Off-diagonal blue values suggest well-predicted interactions between domains. (G) Superposition of the C α -traces of the experimental structures of promirolysin (PS in sienna, CD in gold) and proulilysin (cyan/dodger blue) in standard orientation [ 15 ] onto the prediction of CPL1 (purple/pink). The CPL1 prediction matches proulilysin significantly better. The catalytic zinc (magenta sphere) and the common calcium (red sphere) of proulilysin are further displayed. With respect to the relative orientations of the domains, only the first five domains consistently appeared in a comparable arrangement in the five predictions (see Figure 1E). This is consistent with the fact that relative orientations of domains cannot usually be predicted accurately [ 57 ]. Indeed, analysis of the predicted aligned error, which accounts for the residue–residue alignment confidence and, thus, if domains are reliably positioned [ 58 ], suggests that PS–CD, CD–D3, as well as D3–D4 and D4–D5 might be correctly placed relative to one another in the predictions (Figure 1F). Visual inspection and computational quantification of the inter-domain interfaces with Pisa [ 46 ] suggested that the relative arrangement of PS–CD and CD–D3 might actually be significant. It features interfaces spanning 1329 Å 2 and 518 Å 2 , respectively, which are in the range of values reported for experimental protein–protein complexes (955 ± 380 Å 2 ) [ 59 ], and the calculated ∆i G values are − 12.2 kcal/mol and − 8.9 kcal/mol, respectively. In contrast, the D3–D4 and D4–D5 interfaces feature only few contacts and may rather represent hinge points for structure rearrangement. Future studies may include multi-state small-angle X-ray scattering (SAXS) profile analysis of near-to-full-length protein preparations to experimentally capture the structural interplay of accessory domains. 3.2. Comparison with Proulilysin and Promirolysin Superposition of the experimental LOPAP structure of Tannerella promirolysin (Protein Data Bank [PDB] access code 6R7V [ 23 ]) onto the CPL1 working model revealed 213 aligned residues (out of 307 of promirolysin), with a core rmsd of 2.7 Å and a sequence identity of 30%. The same calculations employing Methanosarcina proulilysin (PDB 8CD8 [ 21 ]) revealed 248 aligned residues (out of 306 proulilysin residues) deviating 1.7 Å (29% identity). Overall, the three structures accurately match and share the common structural elements of LOPAPS (Figures 1A,G and 2A). However, the differences in the rmsd values point to CPL1 being closer to ulilysin than to mirolysin. In all structures, the PS runs across the cleft of the CD in the opposite direction of a substrate, which prevents autolytic cleavage, and encompasses two large, characteristic helices ( α 1p and α 2p). This segment blocks access of substrates to the active-site cleft, thus keeping the zymogens inactive. The helices deviate in the Tannerella zymogen when compared with the other two structures, which accurately match (Figure 1F). Moreover, C 24 of CPL1’s PS would be topologically equivalent to C 23 of both mirolysin and ulilysin (other proteins’ positions are numbered in subscript), which have been shown to play a role in latency in these enzymes by acting as a “cysteine switch” [ 21 , 23 ]. The cysteine S γ atom blocks the catalytic zinc ion as also described for other metzincins, like matrix metallopeptidases, bacterial astacins, adamalysins, and a-disintegrin-and-metallopeptidase enzymes (ADAMs) [ 60 – 63 ]. Furthermore, the experimental activation cleavage site of CPL1 (A 66 –E 67 ) [ 32 , 33 ], which removes the PS and yields the mature, competent CD (Figure 1A,G), would be topologically equivalent to those of the archaeal (S 60 –R 61 ) and bacterial (S 54 –R 55 ) relatives [21,23]. Biomolecules 2024,14, 1604 8 of 20 Biomolecules 2024, 14, x FOR PEER REVIEW 9 of 20 Figure 2. Structural analysis of the predicted CPL1 domains. (A) Ribbon-type plot of the CPL1 PS and CD in cross-eye stereo. The secondary structure elements are labelled (α1p, α2p, α1–α9, and β1–β6). The putative cysteine-switch cysteine (C24), zinc-binding residues (H231, H235 and H241), general base/acid glutamate (E232), Met-turn methionine (M284) and tyrosine-switch tyrosine (Y286), calcium-binding residues (D251 and T256), as well as the putative disulfide-bonded cysteines (C247–C273;  and C267–C292; ) are shown for their side chains as sticks and numbered. The zinc and calcium cations were modelled based on the proulilysin (PDB 8CDB) and mature ulilysin (PDB 2CKI) structures. The putative maturation site (A66–E67) and the LNR-loop are highlighted by green and orange arrows, respectively. Depiction of the Ig-like domains (D3–D10) showing as ribbonor Cα-plots (B) D3; (C) D5 (cyan Cα-plot) onto D3 (plum Cα-plot) in the same orientation as in (B); (D) D4; (E) D6 (brown Cα-plot) onto D4 (yellow Cα-plot) in the same orientation as (D); (F) D7; (G) D9 (orange Cαplot) onto D7 (green Cα-plot) in the same orientation as (F); (H) D8 (disulfide bond C963–C1083; ) and (I) D10. The β-strands and the Nand C-terminal residues are numbered in all cases. 3.3. Eight C-Terminal Immunoglobulin-Like Domains (D3–D10) Downstream of the CD, domain D3 would adopt a compact immunoglobulin-like (Ig-like) fold [67–69], which consists of 117 residues forming an antiparallel β-sandwich with four-stranded front and back sheets (β1–β3–β6–β5 and β2–β8–β7–β4, respectively, from left to right in Figure 2B), with an intersheet angle of ~35° and Greek-key topology. Figure 2. Structural analysis of the predicted CPL1 domains. (A) Ribbon-type plot of the CPL1 PS and CD in cross-eye stereo. The secondary structure elements are labelled ( α 1p, α 2p, α 1– α 9, and β 1– β 6). The putative cysteine-switch cysteine (C 24 ), zinc-binding residues (H 231 , H 235 and H 241 ), general base/acid glutamate (E 232 ), Met-turn methionine (M 284 ) and tyrosine-switch tyrosine (Y 286 ), calcium-binding residues (D 251 and T 256 ), as well as the putative disulfide-bonded cysteines (C 247 –C 273 ; 1  and C 267 –C 292 ; 2  ) are shown for their side chains as sticks and numbered. The zinc and calcium cations were modelled based on the proulilysin (PDB 8CDB) and mature ulilysin (PDB 2CKI) structures. The putative maturation site (A 66 –E 67 ) and the LNR-loop are highlighted by green and orange arrows, respectively. Depiction of the Ig-like domains (D3–D10) showing as ribbonor C α -plots (B) D3; (C) D5 (cyan C α -plot) onto D3 (plum C α -plot) in the same orientation as in (B); (D) D4; (E) D6 (brown C α -plot) onto D4 (yellow C α -plot) in the same orientation as (D); (F) D7; (G) D9 (orange C α -plot) onto D7 (green C α -plot) in the same orientation as (F); (H) D8 (disulfide bond C 963 –C 1083 ; 1  ) and (I) D10. The β -strands and the Nand C-terminal residues are numbered in all cases. As to the CD (Figures 1G and 2A), CPL1 would share with the two LOPAPs the five-stranded β -sheet (top to bottom, β 2+ β 3– β 4– β 6– β 5 in CPL1) including the “LNR-like loop” (L 131 –G 143 ), which protrudes from the molecular surface and divides the second Biomolecules 2024,14, 1604 9 of 20 strand of the sheet in two ( β 2+ β 3). Next, the active-site helix ( α 6) and the backing helix ( α 1), as well as the two short helices ( α 2 and α 3) after the latter, which form a cape in the back of the molecule, would be shared. Further common elements are the Met-turn, the C-terminal helix ( α 8), and two additional α -helices ( α 8 and α 9) after the C-terminal helix, which are characteristic of LOPAPs but not of metzincins in general. Moreover, the active site centred on the catalytic zinc ion and the arrangement of the residues encompassing the extended zinc-binding motif would be very similar, which further supports that CPL1 is actually an MP (Figure 2A) (see also Section 3.5). In addition, the predicted CD disulfides C 247 –C 273 and C 267 –C 292 would be topologically equivalent to C 250 –C 277 and C 269 –C 297 of ulilysin and C 243 –C 271 and C 262 –C 291 of mirolysin. Furthermore, Y 286 (promirolysin) and Y 292 (proulilysin), which are found two positions downstream of the Met-turn methionine (Figure 1A), have been reported to play a role in substrate binding during catalysis, acting as a “tyrosine switch” [ 10 ]. This residue would also be present in CPL1 as Y 286 (Figure 2A), likely exerting similar functions. Finally, similarly to proulilysin, the CPL1 zymogen would lack one of two calcium-binding sites, which exert essential structural functions in the mature structures [ 21 – 25 ]. In contrast, this calcium site is already present in the promirolysin zymogen [ 23 ]. As to the second site, the model adopts a very similar trajectory to the experimental structures for the protein chain involved (D 251 –T 256 ; Figure 1G), thus supporting this site would also be present in CPL1. Unique for CPL1, an “adamalysin helix” ( α 4) would be inserted between β 3 and β 4, nestling on the convex face of the β -sheet (Figure 2A). Such a helix was first reported for adamalysins/ADAMs [ 64 ] and then for fragilysin-3 [ 65 , 66 ], and it is replaced in ulilysin and mirolysin by an irregular loop [ 22 , 23 ]. Moreover, an additional helix ( α 5), which is missing in the other LOPAPs, would be inserted between sheet strands β5 and β6. 3.3. Eight C-Terminal Immunoglobulin-like Domains (D3–D10) Downstream of the CD, domain D3 would adopt a compact immunoglobulin-like (Iglike) fold [67–69], which consists of 117 residues forming an antiparallel β-sandwich with four-stranded front and back sheets ( β 1– β 3– β 6– β 5 and β 2– β 8– β 7– β 4, respectively, from left to right in Figure 2B), with an intersheet angle of ~35 ◦ and Greek-key topology. The predicted model of the domain after next (D5) encompasses 120 residues and superposes very accurately onto D3, with 114 aligned residues overlapping with a core rmsd of 1.2 Å (Figure 2C). The two domains have 37% sequence identity, so they might be functionally equivalent. Notably, Ig-like domains also represent an intriguing scaffold for the de novo design of antibody-like structures with superior biophysical properties [70,71]. With respect to function, D3 and D5 best match domain Ig-2 of the high-molecularmass chitinase ChiW from Paenobacillus sp. (rmsd 3.0 Å; Z-score 9.1 according to [ 48 ]; PDB 5GZT [ 72 ]). Like D3, this domain is also immediately downstream of and attached to a CD, in this case a carbohydrate hydrolase moiety. Both models share the overall architecture, topology, and connectivity, but not the detailed chain trace, as revealed by the rather high rmsd value. Domain Ig-2 and others alike have been proposed to be linkers that connect CDs—or these with substrate-binding domains—and stabilize them within large multimodular enzymes [ 72 ]. The function of D3, and by extension D5, in CPL1 could be similar. Indeed, in the probably most reliably predicted relative arrangement between domains of the working model (see Section 3.1), D3 would interact through its lateral sandwich surface framed by strands β 2 and β 8 with the LNR-like loop of the preceding CD. This interaction would be cemented through a disulfide bond (C140–C434) that is unique for LOPAPs. Linked to D3, the 147-residue D4 domain would likewise be an antiparallel 4+4 β - sandwich (Figure 2D) with a front sheet, in which the first strand is halved by a bulge (from left to right, β 6– β 7– β 8– β 1a+ β 1b), and a back sheet ( β 5– β 4– β 3– β 2). This domain differs from D3/D5 in the topology of the β -sheets. In addition, the difference in size is accounted for by an 11-residue C-terminal extension and a wide 41-residue spiral segment, which connects strands β4 and β5 and laterally contacts the left sandwich surface framed by strands β 5 and β 6 (Figure 2D). 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