Dissecting capture and twisting of aureolysin and pseudolysin: functional amino acids of the Dispase autolysis-inducing protein

The Dispase autolysis-inducing protein (DAIP) from Streptomyces mobaraensis attracts M4 metalloproteases, which results in inhibition and autolysis of bacillolysin (BL) and thermolysin (TL). The present study shows that aureolysin (AL) from Staphylococcus aureus and pseudolysin (LasB) from Pseudomonas aeruginosa are likewise impaired by DAIP. Complete inhibition occurred when DAIP significantly exceeded the amount of the target protease. At low DAIP concentrations, AL and BL performed autolysis, while LasB and TL degradation required reductants or detergents that break intramolecular disulfide bonds or change the protein structure. Site directed mutagenesis of DAIP and removal of an exposed protein loop either influenced binding or inhibition of AL and TL but had no effect on LasB and BL. The Y170A and Δ 239-248 variants had completely lost affinity for TL and AL. The exchange of Asn-275 also impaired the interaction of DAIP with AL. In contrast, DAIP Phe-297 substitution abolished inhibition and autolysis of both target proteases but still allowed complex formation. Our results give rise to the conclusion that other, yet unknown DAIP amino acids inactivate LasB and BL. Obviously, various bacteria in the same habitat caused Streptomyces mobaraensis to continuously optimize DAIP in inactivating the tackling metalloproteases.


Introduction
Staphylococcus aureus and Pseudomonas aeruginosa are human pathogens causing severe infections such as pneumonia or cystic fibrosis [1,2]. Many other diseases are associated with both bacteria that may be especially fatal for hospitalized patients due to the wide distribution of antibiotic-resistant clinical strains [3]. A series of virulence factors have been recognized, among them metalloproteases of the thermolysin family M4, which are thought to play a major role in subverting the defence systems of the host [4]. Aureolysin (AL,EC 3.4.25.29, UniProt P81177, PDB 1BQB), the metalloprotease from S. aureus, contributes to immunoglobulin degradation by activation of the endogenous cysteine protease V8 [5], blood coagulation by prothrombin cleavage [6], septicaemia or staphylococcal immune evasion by inactivation of the α 1 -antichymotrypsin, α 1 -proteinase and antimicrobial LL-37 peptide inhibitors [7][8][9]. It further modulates T and B lymphocytes [10], the complement system [11], the production of immunoglobulins [10], and the killing of phagocytosed bacterial cells [12]. Pseudomonal elastase (pseudolysin, LasB, EC 3.4.24.26, UniProt P14756, PDB 1EZM) is the major secreted protease of P. aeruginosa and an acknowledged virulence factor in animal infection models due to its ability of tissue destruction and escape from the host immune system [13,14]. Targets of LasB are many proteins such as elastin [15] Metalloproteases of the thermolysin (M4) family exhibit a multi-domain structure, commonly consisting of fungalysin/thermolysin propeptide (FTP), PepSY propeptide, N-terminal β-sheet (NTD), and C-terminal α-helix domains (CTD) ( Figure S1A) [32]. The structure of few variants such as the transglutaminase-activating metalloprotease is completed by an additional convertase domain following CTD [33]. While FTP/PepSY act as internal chaperone supporting the protease to adopt its functional structure, NTD and CTD accommodate the active site amino acids comprising the canonical HEXXH motif and a typically 20aa-remote glutamate [34]. FTP and PepSY inhibit NTD and CTD by Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200407/887277/bcj-2020-0407.pdf by guest on 07 July 2020 4 crosswise interaction during folding, thus directing the cleavable peptide bond between propeptide and mature domains to the active site such that auto-catalytical activation can take place. Mature thermolysin is stabilized by four bound calcium ions, referred to as Ca(1-4), which contribute to the extraordinary heat-resistance of the protease ( Figure S1A) [35]. Interestingly, removal of a single calcium ion, Ca(3), in the N-terminal β-sheet domain NTD triggers autolysis of thermolysin [36]. In contrast, aureolysin is not enabled to bind Ca(3), and pseudolysin is only equipped with a single calcium ion in proximity to the active site [37,38]. Stability of pseudolysin is ensured by two disulphide bonds, which are absent in the structures of thermolysin and aureolysin. One pseudolysin cross-bridge seems to substitute the missing Ca(3) in the NTD while the other link the C-terminal peptide to the penultimate helix of the CTD ( Figure S1B).
Thermolysin has been extensively used as zinc-dependent model protease for the design of sitedirected inhibitors, especially due to its close relationship to human angiotensin-converting enzyme and carboxypeptidase A [39]. More than 200 x-ray structures are recorded in the PDB database shedding light on interaction between thermolysin and synthetic hydroxamates [40], carboxylates [41], phosphonamidates [42,43], or mercaptans [44]. Crystal structures of pseudolysin inhibited by a synthetic carboxylate and phosphoramidon, the natural phosphoramidate from Streptomyces tanashiensis [45], are reported (PDB 1U4G, 3DBK) as well. Moreover, new approaches were developed regarding the inhibition of pseudolysin as expedient strategy for treating bacterial infections caused by P. aeruginosa [46][47][48].
We have discovered the Dispase autolysis-inducing protein (DAIP, UniProt P84908, PDB 5FZP) from Streptomyces mobaraensis that binds M4 metalloproteases with high affinity [49,50]. The interaction of DAIP with bacillolysin (BL, Dispase, Gentlyase, EC 3.4.24.28, UniProt P29148, PDB 4GER) results in the immediate self-degradation of the bound metalloprotease while the more resistant thermolysin is only inhibited by DAIP [50]. DAIP adopts a regular seven-bladed beta-propeller, inappropriate of hydrolysing peptide bonds due to the absence of a fused catalytic domain or respective functional amino acids [51]. The rigid beta-propeller of DAIP is thought to modify the bound metalloprotease in such a way that autolysis-prone peptide bonds are exposed, and the active site is twisted [50]. Action of DAIP particularly affects the α-helical domain of metalloproteases as the Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200407/887277/bcj-2020-0407.pdf by guest on 07 July 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200407 crystallized structure of a C-terminal thermolysin fragment in complex with DAIP (PDB 6FHP) suggests [50]. Here, we studied capture, inhibition and autolysis of aureolysin and pseudolysin to show versatility of DAIP in tackling clinical pathogens. Point mutations and removal of an exposed loop, derived from the DAIP thermolysin CTD complex, further illuminate the role of DAIP in attraction and twisting of M4 metalloproteases.
The production of aureolysin (AL) was performed according to the procedure of Nickerson et al. [52]. In brief, a codon-optimized gene (Genscript) encoding His 6 -proaureolysin in pET22b(+) (Merck, Darmstadt, Germany) was used to transform E. coli BL21(DE3)RIL (Agilent Technologies, Waldbronn, Germany). Upon culture in auto-induction medium (10 g/l pepton, 5 g/l yeast extract, 10 g/l NaCl, 0.45 g/l glucose, 4.5 g/l glycerol, 1.2 g/l lactose, 22 mM KH 2 PO 4 , 17.3 mM Na 2 HPO 4 , 1.8 mM MgSO 4 ) for 22 h at 28 °C, the supernatant of 6 l disrupted cells in 50 mM phosphate, 500 mM NaCl, and 6 M urea pH 7.4 was applied onto a 5 ml His Trap HP column (GE Healthcare, Darmstadt, Germany) using a flow rate of 5 ml/min. Protein folding, self-activation and elution of active AL was then induced at flow rates of 3 ml/min by linear decreasing urea from 6 M to 1 M (50 min) and from 1 M to 0 M (15 min). The combined AL fractions were concentrated by acetone precipitation (4 °C, 2.5 volumes of -20 °C solvent) and Fractogel EMD TMAE (bed volume of 3 ml, Merck) binding in 20 mM 5 mM CaCl 2 and Tris/HCl pH 7.5 containing 0 M (AL binding) or 1 M NaCl (AL elution).
Modification of DAIP was carried out by size overlap extension PCR following standard protocols.
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Protease assays
The activity of the metalloproteases was determined according the method of Weimer et al. [53]. In brief, intrinsically quenched dabSFans (39.2 µM) in 2 mM CaCl 2 and 50 mM Tris/HCl pH 7.5 was hydrolyzed by 1.2 µM metalloprotease at 30 °C (final volume of 200 µl). Fluorescence of cleaved Phe-EDANS was continuously monitored at 520 nm (λ exc of 340 nm).
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Other procedures
Determination of protein content by bicinchoninic assay and protein melting points was performed as described previously [50,51].

Production of the proteins
The crystal structure of DAIP in complex with the C-terminal thermolysin fragment [50] was used to uncover functional amino acids that are involved in binding and twisting of M4 metalloproteases.
coli according to our published procedures for bacillolysin (BL) and E138A-BL [50] to further examine DAIP-mediated inactivation of metalloproteases from pathogens. The preceding signal peptides allowed secretion into the periplasmic space, and a C-terminal oligohistidine tag enabled the purification by IMAC. The preparation of non-functional proteases required the split production of pro-enzyme and non-functional mature domains [50] and, upon metal affinity chromatography, degradation of the pro-peptide by trypsin that did not impair the mature protease domains NTD and CTD in general. Other purification steps were acetone precipitation, Fractogel EMD TMAE, phenyl-Sepharose, Gly-DPhe affinity or size exclusion chromatography. While highly purified E141A-LasB, TL, and E143A-TL were obtained in excellent yields ( Figure S2), the amount of active AL remained low, only indicated by slow hydrolysis of the dabSFans dipeptide. A strong protein band at 60 kDa indicated presence of the unprocessed precursor molecule as result of misfolding and failed selfactivation.
We then decided to reproduce the intra-cellular production procedure of Nickerson and colleagues comprising urea-mediated dissolution of His 6 -pro-AL and folding on immobilized metal affinity resins [52]. AL then adopts the functional structure by reduction of urea and detaches from the metal-bound pro-peptide by auto-activation. Although yields were slightly enhanced by this procedure, mature AL Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200407/887277/bcj-2020-0407.pdf by guest on 07 July 2020 8 was strongly contaminated by other proteins, most likely by large amounts of AL fragments ( Figure   S3). Any further purification was associated with severe loss of the protease, even revealing significance of Ca(3) and cysteine cross-bridges in TL and LasB stabilisation that are absent in AL.
The respective IMAC fractions of AL were combined, concentrated by acetone precipitation and DEAE resin binding, and stored at -80 °C.

Capture of M4 metalloproteases by DAIP
We next studied the interaction of the produced metalloproteases with DAIP. Active and nonfunctional bacillolysin, BL or E138A-BL, were used as controls. Size exclusion chromatography indicated high affinity of DAIP for each metalloprotease by the reduced retention times of binary complexes and the complete disappearance of single proteins at equimolar concentrations ( Figure 1A).
The strong interaction of DAIP with the metalloproteases was confirmed by isothermal titration calorimetry (ITC) (Figure 2). While, like BL and E138A-BL, binding of E141A-LasB by DAIP was an endothermic process driven by an increase in entropy, the capture of recombinant TL enhanced enthalpy and entropy as was already shown for the commercial thermolysin [50]. Low dissociation constants of 5-50 nM revealed the detection limit of ITC. As reported earlier [50], DAIP binds two bacillolysin molecules under ITC conditions, which we believe that they form a piggyback complex, one protease molecule in complex with DAIP, the other in complex with the bound protease molecule.
It should be further noted that the obtained amounts of active aureolysin were too small to perform similar SEC or ITC experiments.
The metalloproteases E138A-BL and TL were further studied by CD spectroscopy to observe structural modification by DAIP-mediated complex formation ( Figure 1B). The DAIP ellipticity spectrum was characterized by a broad minimum at 215 nm as may be expected for propeller proteins shaped by β-strands (blue dots). The NTD (β-strands) and CTD domains (α-helices) of both metalloproteases caused two distinct minima around 210 nm and 220 nm (red dots) that almost disappeared in the equimolar complex with DAIP (green dots). A comparison of added individual and complex spectra (green and purple dots) only showed modified ellipticity between 195 nm and 198 nm, thus suggesting that few structural elements were changed in the DAIP complex of E138A-BL Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200407/887277/bcj-2020-0407.pdf by guest on 07 July 2020 9 and TL. As β-propellers usually possess a rigid structure, changes in the metalloprotease structures appear to be more likely.

DAIP-mediated inhibition and autolysis of M4 metalloproteases
We then compared inhibition and autolysis of the metalloproteases as a function of DAIP concentration. The most remarkable result was that autolysis of bacillolysin (BL), the most susceptible metalloprotease, was completely inhibited by high amounts of DAIP ( Figure 3A). In other words, at high concentrations, DAIP alters BL in such a way that autoproteolytic degradation cannot take place.
Absence of proteolytic activity was either observed by the persistence of the BL electrophoresis band ( Figure S4A) but, more importantly, by the absence of BL fragments, which increase SYPRO orange fluorescence [50]. A similar result was obtained with active AL, TL and LasB ( Figure 3B-D, Figure   S4B-D). However, it should be noted that inhibited TL still allowed dabSFans access to the active site, thus keeping residual activity of about 10 percent ( Figure 3C). The inhibition of all studied metalloproteases substantiates our former conclusion [50] that DAIP most likely changes the structure near the active site of the proteolytic enzymes.
Complete reduction of BL activity prompted us to investigate the effect of preincubation on DAIP-mediated inhibition and autolysis. Preincubation of BL with DAIP for 30 min resulted in rapid decomposition of BL if the amount of DAIP was too small to inhibit the protease completely. Hence, the effective concentration allowing DAIP to inactivate BL by half (EC 50 ) was correspondingly reduced by one order of magnitude ( Figure 3A). A similar result was obtained with aureolysin ( Figure   3B). In contrast, as LasB and TL resisted autolysis within the analyzed time span, the EC 50 of DAIP were not affected by preincubation ( Figure 3C/D). Former experiments have shown that detergents accelerate the decomposition of thermolysin by DAIP [50]. Similarly, the reduction of intramolecular disulfide bonds, lacking in BL, AL, and TL, caused pseudolysin to perform autolysis in the presence of DAIP ( Figure S5).

Functional amino acids of DAIP influencing capture and twisting of M4 metalloproteases
The CTD fragment of thermolysin (from Gly-257 to Val-315) in complex with DAIP (PDB 6FHP) superposed well with full-length TL (PDB 2TLX), but the TL loop region 115 WNGS 118 and amino Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200407/887277/bcj-2020-0407.pdf by guest on 07 July 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200407 acids in close vicinity severely clash with DAIP ( Figure 4A/B, red box) [50]. Importantly, access of protease substrate molecules seems to be possible as the TL active site is not covered by DAIP. The nearly unchanged structure of DAIP suggests that the reduced activity of TL is the result of active site twisting. Especially, Phe-297 of the clashing DAIP β-turn seems to collide with β-sheet 9 and αhelix 2 of thermolysin that accommodates the Zn 2+ -binding Glu-143 and His-146 of the active site.
( Figure 4C, Figure S6, Figure S7). Moreover, several DAIP amino acids in close contact with thermolysin or the thermolysin CTD fragment were considered excellent candidates for site-directed mutagenesis ( Figure 4D).
Initially, binding sites for two Ca 2+ ions, which cross-link two DAIP molecules in the crystal (PDB 5FZP), were substituted by alanine. However, substitution of Glu-247, Asp-267, Glu-272, and Glu-343 had no effect on DAIP activity ( Figure S6). Only AL inhibition was impaired by the exchange of Asp-322 (Table 1, Figure 4C/D). The result was consistent with earlier observations that transfer of proteinase-stabilizing calcium ions is not the reason for autolysis [49].
Next, the prominent loop between Glu-239 and Gly-248 of DAIP (Δ 239-248 ) was removed, and the clashing Phe-297 within the rigid β-turn between Thr-295 and Gly-301 was exchanged for alanine  (Table 1). Indeed, none of the introduced mutations could decisively weaken DAIP-mediated capture and inhibition in LasB and BL. On the contrary, BL seemed to perform autolysis faster in the presence of the F297A and Δ239-248 variants. In any case, the introduced mutations clearly discriminated between DAIP amino acids that either contribute to the attachment of aureolysin and thermolysin or to conformational changes, which affect the proteolytic activity. Absence of heat release, depicted by ITC, suggested that substitution of Tyr-170 completely abolishes the ability of DAIP to capture both metalloproteases. AL and TL inhibition was likewise disrupted by the replacement of Tyr-170 (Table 1, Figure 5). Furthermore, the Δ 239-248 loop of DAIP seems to act like a "bumping post" or an orientation guide for the proper docking of the target metalloprotease. Erasing Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200407/887277/bcj-2020-0407.pdf by guest on 07 July 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200407 the loop impeded the attachment of thermolysin to DAIP that considerably higher amounts of DAIP (more than two orders of magnitude) were required to reduce the proteolytic activity. Loop removal was still more effective in aureolysin as proteolytic activity remained unchanged in the presence of Δ 239-248 -DAIP. Erasing DAIP activity for aureolysin by Asn-275 substitution further proved that specific amino acids at the DAIP surface might individually control the attachment of a target protease ( Figure 5).
The amino acid Phe-297 within the prominent β-turn of DAIP works completely different from Tyr-170, Asn-275 and the loop between Glu-239 and Gly-248. Removal of the aromatic ring in F297A further allowed DAIP to attract thermolysin and aureolysin but abolished completely the inhibitory activity ( Figure 5). The hydrolysis rate of TL was even considerably enhanced by the F297A variant, thus shedding light on the β-turn of DAIP. This β-turn protrudes the propeller surface like a thorn and includes between Gly-294 and Gly-301 ( 294 GTYFQAYG 301 ) several space-demanding residues. While the side-chain of Phe-297 impairs activity of thermolysin and aureolysin, other bulky DAIP amino acids such as Tyr-296, Tyr-300, Gln-298, and even the methyl group of Ala-299 may influence twisting and autolysis of other metalloproteases. The close vicinity to the β-turn and the involvement in AL inhibition further suggest that Asp-322 could contribute to the structural changes. In any case, amino acids at the upper surface of the rigid DAIP propeller have two different functions, both target molecule binding and structure transformation, that seem to be exerted independently but in a cooperative manner. Specific, even not yet identified, capture amino acids could provide for the destruction of a large number of harmful metalloproteases in the habitat of S. mobaraensis.
The study confirms the recently postulated three-step mechanism for the DAIP catalysis consisting of (1) capture of the M4 metalloproteases, (2) inhibition as result of twisting, and (3) autolysis [50]. Protein association and twisting are obviously separate processes, albeit functional amino acids influencing subsistence of bacillolysin and pseudolysin remain to be determined.

Conclusions
The Dispase autolysis-inducing protein (DAIP) is a powerful tool to control proteolytic activity of neutral M4 metalloproteases in the habitat of Streptomyces mobaraensis. As shown here, aureolysin Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200407/887277/bcj-2020-0407.pdf by guest on 07 July 2020 (AL) and pseudolysin (LasB) from the pathogenic bacteria Staphylococcus aureus and Pseudomonas aeruginosa are targets of DAIP as was already proven for bacillolysin (BL) from Paenibacillus polymyxa and thermolysin (TL) from Bacillus thermoproteolyticus rokko [50]. These enzymes are captured and twisted by DAIP in a way that their proteolytic activity is inhibited at low protease concentration. As the amount of target protease increases, unbound (active) protease molecules hydrolyze the autolysis-prone captives in complex with DAIP. Degradation of BL and AL occurs rapidly while TL and LasB need support by detergents or reductants that modify the structure and open the cysteine cross-bridges, respectively. Binding of the target proteases and twisting are mediated by different amino acids of DAIP as was shown here. A DAIP tyrosine, Tyr-170, seems to be essential for the attachment of thermolysin and aureolysin, possibly by π-π-stacking with Tyr-274 or Tyr-261, conserved in TL and AL ( Figure S6, Figure  The continued binding of thermolysin by the F297A variant further showed that capture and inhibition are two independent steps of the DAIP inactivation mechanism. Unfortunately, the introduction of more than single point mutations destabilizes the DAIP structure, thus limiting the investigation of more amino acids or multiply substituted variants. Only structure of the respective metalloprotease in complex with DAIP will reveal the individual hot spots. However, such expedient approach is a challenge. Even the smallest traces of the active protease will degrade the DAIP-modified enzymes during the crystallization procedure.

Declarations of Interest
The authors declare no conflict of interest.

Author contribution
DF, HLF designed research; DF, SAD performed research; DF, SAD, AA, HK, HLF analysed data; DF, HLF wrote the paper with comments from HK.   b) Upon mixing various concentrations of DAIP with 50 nM BL, 5 nM TL, 4.5 nM AL or 6.3 nM LasB in 50 mM Tris, 5 mM CaCl 2 pH 8.0, proteolytic activity was immediately measured using dabSFans. The data are the mean of three independent measurements. -c) The used aureolysin, was highly contaminated, most likely by AL fragments.

References
Downloaded from https://portlandpress.com/biochemj/article-pdf/doi/10.1042/BCJ20200407/887277/bcj-2020-0407.pdf by guest on 07 July 2020 Biochemical Journal. This is an Accepted Manuscript. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date-version is available at https://doi.org/10.1042/BCJ20200407    Mixtures of 50 nM bacillolysin (A), ~4.5 nM aureolysin (B), 5 nM thermolysin (C), and 6.5 nM pseudolysin (D) and the indicated molar ratio of DAIP in 50 mM Tris pH 7.5 and 2 mM CaCl 2 were preincubated (green line) for 30 min at 37 °C or not (blue line). Residual activity was determined in triplicates by hydrolysis of dabSFans and increase in fluorescence at 520 nm (λ exc of 340 nm).