Decoding the Papain Inhibitor from Streptomyces mobaraensis as Being Hydroxylated Chymostatin Derivatives: Purification, Structure Analysis, and Putative Biosynthetic Pathway.

Streptomyces mobaraensis produces the papain inhibitor SPI consisting of a 12 kDa protein and small active compounds (SPIac). Purification of the papain inhibitory compounds resulted in four diverse chymostatin derivatives that were characterized by NMR and MS analysis. Chymostatins are hydrophobic tetrapeptide aldehydes from streptomycetes, e.g., S. lavendulae and S. hygroscopicus, that reverse chymosin-mediated angiotensin activation and inhibit other serine and cysteine proteases. Chymotrypsin and papain were both inhibited by the SPIac compounds in the low nanomolar range. SPIac differs from the characterized chymostatins by the exchange of phenylalanine for tyrosine. The crystal structure of one of these chymostatin variants confirmed its molecular structure and revealed a S-configured hemithioacetal bond with the catalytic Cys25 thiolate as well as close interactions with hydrophobic S1 and S2 subsite amino acids. A model for chymostatin biosynthesis is provided based on the discovery of clustered genes encoding several putative nonribosomal peptide synthetases; among them, there is the unusual CstF enzyme that accommodates two canonical amino acid activation domains as well as three peptide carrier protein domains.

Mutations in the major gas vesicle protein GvpA and impacts on gas vesicle formation in Haloferax volcanii.

Gas vesicles are proteinaceous, gas-filled nanostructures produced by some bacteria and archaea. The hydrophobic major structural protein GvpA forms the ribbed gas vesicle wall. An in-silico 3D-model of GvpA of the predicted coil-α1-ß1-ß2-α2-coil structure is available and implies that the two ß-chains constitute the hydrophobic interior surface of the gas vesicle wall. To test the importance of individual amino acids in GvpA we performed 85 single substitutions and analyzed these variants in Haloferax volcanii ΔA + Amut transformants for their ability to form gas vesicles (Vac+ phenotype). In most cases, an alanine substitution of a non-polar residue did not abolish gas vesicle formation, but the replacement of single non-polar by charged residues in ß1 or ß2 resulted in Vac- transformants. A replacement of residues near the ß-turn altered the spindle-shape to a cylindrical morphology of the gas vesicles. Vac- transformants were also obtained with alanine substitutions of charged residues of helix α1 suggesting that these amino acids form salt-bridges with another GvpA monomer. In helix α2, only the alanine substitution of His53 or Tyr54, led to Vac- transformants, whereas most other substitutions had no effect. We discuss our results in respect to the GvpA structure and data available from solid-state NMR.

Shedding light on biofilm formation of Halobacterium salinarum R1 by SWATH-LC/MS/MS analysis of planktonic and sessile cells.

Early and mature biofilm formation in the extremely halophilic euryarchaeon Halobacterium salinarum strain R1 was characterized by SWATH-LC/MS/MS. Using a simple surfactant-assisted protein solubilization protocol and one-dimensional ultra-high performance nanoflow chromatography on the front end, 63.2 and 58.6% of the predicted H. salinarum R1 proteome could be detected and quantified, respectively. Analysis of biophysical protein properties, functional analysis and pathway mapping indicated comprehensive characterization of the proteome. Sixty point eight percent of the quantified proteins (or 34.5% of the predicted proteome) exhibited significant abundance changes between planktonic and sessile states, demonstrating that haloarchaeal biofilm formation represents a profound "lifestyle change" on the molecular level. Our results and analysis constitute the first comprehensive study to track molecular changes from planktonic cultures to initial and mature archaeal biofilms on the proteome level. Data are available via ProteomeXchange, identifier PXD003667. Proteins exemplifying different protein expression level profiles were selected, and their corresponding gene transcripts targeted by qRT-PCR to test the feasibility of establishing rapid PCR-based assays for archaeal biofilm formation.

Structure of the Dispase Autolysis-inducing Protein from Streptomyces mobaraensis and Glutamine Cross-linking Sites for Transglutaminase.

Transglutaminase from Streptomyces mobaraensis (MTG) is an important enzyme for cross-linking and modifying proteins. An intrinsic substrate of MTG is the dispase autolysis-inducing protein (DAIP). The amino acid sequence of DAIP contains 5 potential glutamines and 10 lysines for MTG-mediated cross-linking. The aim of the study was to determine the structure and glutamine cross-linking sites of the first physiological MTG substrate. A production procedure was established in Escherichia coli BL21 (DE3) to obtain high yields of recombinant DAIP. DAIP variants were prepared by replacing four of five glutamines for asparagines in various combinations via site-directed mutagenesis. Incorporation of biotin cadaverine revealed a preference of MTG for the DAIP glutamines in the order of Gln-39 ≫ Gln-298 > Gln-345 ∼ Gln-65 ≫ Gln-144. In the structure of DAIP the preferred glutamines do cluster at the top of the seven-bladed ß-propeller. This suggests a targeted cross-linking of DAIP by MTG that may occur after self-assembly in the bacterial cell wall. Based on our biochemical and structural data of the first physiological MTG substrate, we further provide novel insight into determinants of MTG-mediated modification, specificity, and efficiency.

The accessory gas vesicle protein GvpM of haloarchaea and its interaction partners during gas vesicle formation.

Gas vesicles consist predominantly of the hydrophobic GvpA and GvpC, and the accessory proteins GvpF through GvpM are required in minor amounts during formation. GvpM and its putative interaction partners were investigated. GvpM interacted with GvpH, GvpJ and GvpL, but not with GvpG. Interactions were also observed in vivo in Haloferax volcanii transformants using Gvp fusions to the green fluorescent protein smGFP. Cells producing the hydrophobic M(GF)P contained a single fluorescent aggregate per cell, whereas cells containing L(GFP) or H(GFP) were fully fluorescent. The soluble L(GFP) formed stable co-aggregates with GvpM in L(GFP)M transformants, but the presence of GvpH resulted in the absence of M(GF)P foci in HM(GFP) transformants. Substitution- and deletion mutants of GvpM determined functionally important amino acids (aa). Substitution of a polar by a non-polar aa in the N-terminal region of GvpM had no effect, whereas a substitution of a non-polar by a polar aa in this region inhibited gas vesicle formation in transformants. Substitutions in region 44-48 of GvpM strongly reduced the number of gas vesicles, and deletions at the N-terminus resulted in Vac(-) transformants. Gas vesicle morphology was not affected by any mutation, implying that GvpM is required during initial stages of gas vesicle assembly.

The papain inhibitor (SPI) of Streptomyces mobaraensis inhibits bacterial cysteine proteases and is an antagonist of bacterial growth.

A novel papain inhibitory protein (SPI) from Streptomyces mobaraensis was studied to measure its inhibitory effect on bacterial cysteine protease activity (Staphylococcus aureus SspB) and culture supernatants (Porphyromonas gingivalis, Bacillus anthracis). Further, growth of Bacillus anthracis, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio cholerae was completely inhibited by 10 µM SPI. At this concentration of SPI, no cytotoxicity was observed. We conclude that SPI inhibits bacterial virulence factors and has the potential to become a novel therapeutic treatment against a range of unrelated pathogenic bacteria.

Use of GFP-GvpE fusions to quantify the GvpD-mediated reduction of the transcriptional activator GvpE in haloarchaea.

Gas vesicle formation of Halobacterium salinarum is regulated by the transcriptional activator GvpE, and in the presence of the repressing protein GvpD, the amount of GvpE is strongly reduced. The green fluorescence protein was used to report this GvpD-mediated reduction of GvpE in vivo in Haloferax volcanii transformants. Both N- or C-terminal fusions of GFP to GvpE were tested, but only the N-terminal fusion reported the reduction. The fluorescence of GFP-GvpE was 62 % reduced with GvpD wild type (DWT), 78 % with the super-repressor D3-AAA, and only 10 % with the repression defect DMut6. Further analysis of D3-AAA indicated that the super-repression was due to the alteration R496A. GFP-GvpE variants defect in promoter activation was tested in the presence of DWT, D3-AAA and DMut6, and two of them were more stable. Overall, the GFP-GvpE fusion was suitable to study and quantify the amount of GvpE in vivo.

Effect of an overproduction of accessory Gvp proteins on gas vesicle formation in Haloferax volcanii.

Gas vesicle formation in haloarchaea requires the expression of the p-vac region consisting of 14 genes, gvpACNO and gvpDEFGHIJKLM. Expression of gvpFGHIJKLM leads to essential accessory proteins formed in minor amounts. An overexpression of gvpG, gvpH or gvpM in addition to p-vac inhibited gas vesicle formation, whereas large amounts of all other Gvp proteins did not disturb the synthesis. The unbalanced expression and in particular an aggregation of the overproduced Gvp with other accessory Gvp derived from p-vac could be a reason for the inhibition. Western analyses demonstrated that the hydrophobic GvpM (and GvpJ) indeed form multimers. Fluorescent dots of GvpM-GFP were seen in cells in vivo underlining an aggregation of GvpM. In search for proteins neutralizing the inhibitory effect in case of GvpM, p-vac +pGM(ex), +pHM(ex), +pJM(ex), and +pLM(ex) transformants were constructed. The inhibitory effect of GvpM on gas vesicle formation was suppressed by GvpH, GvpJ or GvpL, but not by GvpG. Western analyses demonstrated that pHM(ex) and pJM(ex) transformants contained additional larger protein bands when probed with an antiserum raised against GvpH or GvpJ, implying interactions. The balanced amount of GvpM-GvpH and GvpM-GvpJ appears to be important during gas vesicle genesis.

Structural model of the gas vesicle protein GvpA and analysis of GvpA mutants in vivo.

Gas vesicles are gas-filled protein structures increasing the buoyancy of cells. The gas vesicle envelope is mainly constituted by the 8 kDa protein GvpA forming a wall with a water excluding inner surface. A structure of GvpA is not available; recent solid-state NMR results suggest a coil-α-ß-ß-α-coil fold. We obtained a first structural model of GvpA by high-performance de novo modelling. Attenuated total reflection (ATR)-Fourier transform infrared spectroscopy (FTIR) supported this structure. A dimer of GvpA was derived that could explain the formation of the protein monolayer in the gas vesicle wall. The hydrophobic inner surface is mainly constituted by anti-parallel ß-strands. The proposed structure allows the pinpointing of contact sites that were mutated and tested for the ability to form gas vesicles in haloarchaea. Mutations in α-helix I and α-helix II, but also in the ß-turn affected the gas vesicle formation, whereas other alterations had no effect. All mutants supported the structural features deduced from the model. The proposed GvpA dimers allow the formation of a monolayer protein wall, also consistent with protease treatments of isolated gas vesicles.

Biofilm formation by haloarchaea.

A fluorescence-based live-cell adhesion assay was used to examine biofilm formation by 20 different haloarchaea, including species of Halobacterium, Haloferax and Halorubrum, as well as novel natural isolates from an Antarctic salt lake. Thirteen of the 20 tested strains significantly adhered (P-value < 0.05) to a plastic surface. Examination of adherent cell layers on glass surfaces by differential interference contrast, fluorescence and confocal microscopy showed two types of biofilm structures. Carpet-like, multi-layered biofilms containing micro- and macrocolonies (up to 50 µm in height) were formed by strains of Halobacterium salinarum and the Antarctic isolate t-ADL strain DL24. The second type of biofilm, characterized by large aggregates of cells adhering to surfaces, was formed by Haloferax volcanii DSM 3757T and Halorubrum lacusprofundi DL28. Staining of the biofilms formed by the strongly adhesive haloarchaeal strains revealed the presence of extracellular polymers, such as eDNA and glycoconjugates, substances previously shown to stabilize bacterial biofilms. For Hbt. salinarum DSM 3754T and Hfx. volcanii DSM 3757T , cells adhered within 1 day of culture and remained viable for at least 2 months in mature biofilms. Adherent cells of Hbt. salinarum DSM 3754T showed several types of cellular appendages that could be involved in the initial attachment. Our results show that biofilm formation occurs in a surprisingly wide variety of haloarchaeal species.

A dual promoter region with overlapping activator sequences drives the expression of gas vesicle protein genes in haloarchaea.

Gas vesicle formation in haloarchaea involves 14 gas vesicle protein (gvp) genes. The strong promoter P(A) drives the expression of gvpACNO, which encodes the major gas vesicle structural proteins GvpA and GvpC, whereas the oppositely oriented promoter P(D) initiates the synthesis of the two regulator proteins, GvpD and GvpE. GvpE activates P(A) and P(D), and requires a 20 nt upstream activator sequence (UAS). UAS(A) and UAS(D) partially overlap in the centre of the 35 bp intergenic region. The basal and GvpE-induced activities of P(A) and P(D) were investigated in Haloferax volcanii transformants. Each UAS consists of two 8 nt portions (P(A), 1A+2A; P(D), 1D+2D), and mutations in the overlapping 1A and 1D portions affected the GvpE induction of both promoters. Substitution of one of the UAS portions by a nonsense sequence showed that a complete UAS is required for activation. The activation of P(A) was more efficient compared with P(D). Promoter P(A) with UAS(A) in configuration 1A+1A was still activated by GvpE, but P(D) was not inducible with UAS(D) in configuration 1D+1D. The TATA box and/or transcription factor B recognition element (BRE) were exchanged between P(A) and P(D). All elements of P(A) functioned well in the environment of 'P(D)' and transferred the stronger P(A) activity to 'P(D)'. In contrast, the respective 'P(A)' chimeras were less active, and BRE(D) was not functional in the environment of 'P(A)'. The relative strengths of the two promoters were substantially determined by the BRE. A 4 nt scanning mutagenesis uncovered an additional regulatory element in the region between TATA(D) and the transcriptional start site of gvpD.

Expression of multiple tfb genes in different Halobacterium salinarum strains and interaction of TFB with transcriptional activator GvpE.

Halobacterium salinarum NRC-1 contains multiple TBP and TFB proteins required for the recruitment of RNA polymerase for transcription initiation. The presence and the expression of genes encoding TFB were investigated in the two Hbt. salinarum strains NRC-1 and PHH1 and the mutant strain PHH4. The plasmid-encoded tfbC and tfbE genes of NRC-1 were lacking in PHH1 and PHH4. The 5'-end of the tfbF transcript was determined and contained a 5'-untranslated region of 39 nucleotides able to form a stem-loop structure. The expression of these tfb genes was studied in cultures growing at 15, 37°C and under heat shock conditions. Cold temperatures reduced growth and except for tfbF also the amounts of all tfb transcripts. However, the formation of gas vesicles increased in PHH1 and NRC-1. Heat shock reduced growth of PHH1 and NRC-1, but PHH4 was not affected. A 100-fold increase in tfbA and tfbB mRNA was observed in PHH1 and PHH4, whereas NRC-1 reduced the amounts of these transcripts and increased the expression of tfbG. All TFB proteins tested were able to interact with the transcription activator GvpE involved in gas vesicle formation that thus is able to recruit TFB to the gvp promoter.

Recent Advances in the Study of Gas Vesicle Proteins and Application of Gas Vesicles in Biomedical Research.

The formation of gas vesicles has been investigated in bacteria and haloarchaea for more than 50 years. These air-filled nanostructures allow cells to stay at a certain height optimal for growth in their watery environment. Several gvp genes are involved and have been studied in Halobacterium salinarum, cyanobacteria, Bacillus megaterium, and Serratia sp. ATCC39006 in more detail. GvpA and GvpC form the gas vesicle shell, and additional Gvp are required as minor structural proteins, chaperones, an ATP-hydrolyzing enzyme, or as gene regulators. We analyzed the Gvp proteins of Hbt. salinarum with respect to their protein-protein interactions, and developed a model for the formation of these nanostructures. Gas vesicles are also used in biomedical research. Since they scatter waves and produce ultrasound contrast, they could serve as novel contrast agent for ultrasound or magnetic resonance imaging. Additionally, gas vesicles were engineered as acoustic biosensors to determine enzyme activities in cells. These applications are based on modifications of the surface protein GvpC that alter the mechanical properties of the gas vesicles. In addition, gas vesicles have been decorated with GvpC proteins fused to peptides of bacterial or viral pathogens and are used as tools for vaccine development.

A Synthetic Riboswitch to Regulate Haloarchaeal Gene Expression.

In recent years, synthetic riboswitches have become increasingly important to construct genetic circuits in all three domains of life. In bacteria, synthetic translational riboswitches are often employed that modulate gene expression by masking the Shine-Dalgarno (SD) sequence in the absence or presence of a cognate ligand. For (halo-)archaeal translation, a SD sequence is not strictly required. The application of synthetic riboswitches in haloarchaea is therefore limited so far, also because of the molar intracellular salt concentrations found in these microbes. In this study, we applied synthetic theophylline-dependent translational riboswitches in the archaeon Haloferax volcanii. The riboswitch variants A through E and E∗ were chosen since they not only mask the SD sequence but also the AUG start codon by forming a secondary structure in the absence of the ligand theophylline. Upon addition of the ligand, the ribosomal binding site and start codon become accessible for translation initiation. Riboswitch E mediated a dose-dependent, up to threefold activation of the bgaH reporter gene expression. Raising the salt concentration of the culture media from 3 to 4 M NaCl resulted in a 12-fold increase in the switching capacity of riboswitch E, and switching activity increased up to 26-fold when the cultivating temperature was reduced from 45 to 30°C. To construct a genetic circuit, riboswitch E was applied to regulate the synthesis of the transcriptional activator GvpE allowing a dose-dependent activation of the mgfp6 reporter gene under P pA promoter control.

Effect of Mutations in GvpJ and GvpM on Gas Vesicle Formation of Halobacterium salinarum.

The two haloarchaeal proteins, GvpM and GvpJ, are homologous to GvpA, the major gas vesicle structural protein. All three are hydrophobic and essential for gas vesicle formation. The effect of mutations in GvpJ and GvpM was studied in Haloferax volcanii transformants by complementing the respective mutated gene with the remaining gvp genes and inspecting the cells for the presence of gas vesicles (Vac+). In case of GvpJ, 56 of 66 substitutions analyzed yielded Vac- ΔJ + Jmut transformants, indicating that GvpJ is very sensitive to alterations, whereas ten of the 38 GvpM variants resulted in Vac- ΔM + Mmut transformants. The variants were also tested by split-GFP for their ability to interact with their partner protein GvpL. Some of the alterations leading to a Vac- phenotype affected the J/L or M/L interaction. Also, the interactions J/A and J/M were studied using fragments to exclude an unspecific aggregation of these hydrophobic proteins. Both fragments of GvpJ interacted with the M1-25 and M60-84 fragments of GvpM, and fragment J1-56 of GvpJ interacted with the N-terminal fragment A1-22 of GvpA. A comparison of the results on the three homologous proteins indicates that despite their relatedness, GvpA, GvpJ, and GvpM have unique features and cannot substitute each other.

Anaerobiosis inhibits gas vesicle formation in halophilic Archaea.

The effect of anaerobiosis on the gas vesicle formation was investigated in three Halobacterium salinarum strains, Haloferax mediterranei and in Haloferax volcanii transformants. All these strains significantly reduced gas vesicle formation or lacked these structures under anoxic conditions. When grown by arginine fermentation, Hbt. salinarum PHH4 lacked gas vesicles, whereas Hbt. salinarum PHH1 and NRC-1 contained 5-20 small gas vesicles arranged in two to three aggregates per cell instead of the 30-80 gas vesicles present under oxic conditions. The enlargement presumably stopped due to a depletion of Gvp proteins. Also Hfx. mediterranei and Hfx. volcanii transformants lacked gas vesicles under anoxic growth and yielded a 10-fold reduced gvp transcription. Even the gas vesicle-overproducing DeltaD transformants did not form gas vesicles under anoxic conditions, demonstrating that the repressing protein GvpD was not involved. The presence of large amounts of GvpA implied that the assembly of the gas vesicles was inhibited. When Hbt. salinarum PHH1 and NRC-1 were grown with dimethyl sulphoxide or trimethylamine N-oxid under anoxic conditions the number but not the size of gas vesicles was reduced. This was in contrast to the previously reported overproduction of gas vesicles in NRC-1 that turned out to depend on the citrate-containing medium used for growth.

Interaction of transcription activator GvpE with TATA-box-binding proteins of Halobacterium salinarum.

GvpE is the transcriptional activator of the gvp gene cluster involved in gas vesicle formation in Haloabacterium salinarum. A 20-nucleotide sequence is required for the GvpE-mediated activation of the two oppositely oriented gvp promoters, P ( A ) and P ( D ). This sequence is located adjacent to the TATA-box and the transcription factor-B-binding site BRE, suggesting an interaction between GvpE and proteins of the transcription initiation apparatus. Here, we analysed the interaction of GvpE with the five different TATA-box-binding proteins, TBP, of Hbt. salinarum PHH1. The His-tagged TbpA through TbpE proteins were produced in Escherichia coli, bound to Ni-NTA matrices and tested for interaction with GvpE by protein-protein affinity chromatography. All Tbp(His) proteins retained the two different GvpE proteins from lysates of Haloferax volcanii transformants expressing the respective gvpE reading frame in pJAS35. Also, both GvpE(His) proteins bound to Ni-NTA matrices retained TbpB, whereas the 20-kDa soluble gas vesicle protein GvpH(His) neither bound TbpB nor GvpE from the respective lysates of Hfx. volcanii. From these results, it appears that GvpE interacts with any TBP of Hbt. salinarum. This interaction might attract TBP and subsequently TFB and RNAP to the promoter and thus enhance transcription of the gvp gene cluster.

Overlapping activator sequences determined for two oppositely oriented promoters in halophilic Archaea.

Transcription of the genomic region involved in gas vesicle formation in Halobacterium salinarum (p-vac) and Haloferax mediterranei (mc-vac) is driven by two divergent promoters, P(A) and P(D), separated by only 35 nt. Both promoters are activated by the transcription activator GvpE which in the case of P(mcA) requires a 20-nt sequence (UAS) consisting of two conserved 8-nt sequence portions located upstream of BRE. Here, we determined the two UAS elements in the promoter region of p-vac by scanning mutageneses using constructs containing P(pD) (without P(pA)) fused to the bgaH reporter gene encoding an enzyme with beta-galactosidase activity, or the dual reporter construct pApD with P(pD) fused to bgaH and P(pA) to an altered version of gvpA. The two UAS elements found exhibited a similar extension and distance to BRE as previously determined for the UAS in P(mcA). Their distal 8-nt portions almost completely overlapped in the centre of P(pD)-P(pA), and mutations in this region negatively affected the GvpE-mediated activation of both promoters. Any alteration of the distance between BRE and UAS resulted in the loss of the GvpE activation, as did a complete substitution of the proximal 8-nt portion, underlining that a close location of UAS and BRE was very important.

Accessory Gvp Proteins Form a Complex During Gas Vesicle Formation of Haloarchaea.

Halobacterium salinarum forms gas vesicles consisting of a protein wall surrounding a gas-filled space. The hydrophobic 8-kDa protein GvpA is the major constituent of the ribbed wall, stabilized by GvpC at the exterior surface. In addition, eight accessory Gvp proteins are involved, encoded by gvpFGHIJKLM that are co-transcribed in early stages of growth. Most of these proteins are essential, but their functions are not yet clear. Here we investigate whether GvpF through GvpM interact. Pull-down experiments performed in Haloferax volcanii with the cellulose-binding-domain as tag suggested many interactions, and most of these were supported by the split-GFP analyses. The latter study indicated that GvpL attracted all other accessory Gvp, and the related GvpF bound besides GvpL also GvpG, GvpH and GvpI. A strong interaction was found between GvpH and GvpI. GvpG showed affinity to GvpF and GvpL, whereas GvpJ, GvpK and GvpM bound GvpL only. Using GvpA for similar analyses yielded GvpF as the only interaction partner. The contact site of GvpF was confined to the N-terminal half of GvpA and subsequently mapped to certain amino acids. Taken together, our results support the idea that the accessory Gvp form a complex early in gas-vesicle assembly attracting GvpA via GvpF.

The N-terminal peptide of the transglutaminase-activating metalloprotease inhibitor from Streptomyces mobaraensis accommodates both inhibition and glutamine cross-linking sites.

Streptomyces mobaraensis is a key player for the industrial production of the protein cross-linking enzyme microbial transglutaminase (MTG). Extra-cellular activation of MTG by the transglutaminase-activating metalloprotease (TAMP) is regulated by the TAMP inhibitory protein SSTI that belongs to the large Streptomyces subtilisin inhibitor (SSI) family. Despite decades of SSI research, the binding site for metalloproteases such as TAMP remained elusive in most of the SSI proteins. Moreover, SSTI is a MTG substrate, and the preferred glutamine residues for SSTI cross-linking are not determined. To address both issues, that is, determination of the TAMP and the MTG glutamine binding sites, SSTI was modified by distinct point mutations as well as elongation or truncation of the N-terminal peptide by six and three residues respectively. Structural integrity of the mutants was verified by the determination of protein melting points and supported by unimpaired subtilisin inhibitory activity. While exchange of single amino acids could not disrupt decisively the SSTI TAMP interaction, the N-terminally shortened variants clearly indicated the highly conserved Leu40-Tyr41 as binding motif for TAMP. Moreover, enzymatic biotinylation revealed that an adjacent glutamine pair, upstream from Leu40-Tyr41 in the SSTI precursor protein, is the preferred binding site of MTG. This extension peptide disturbs the interaction with TAMP. The structure of SSTI was furthermore determined by X-ray crystallography. While no structural data could be obtained for the N-terminal peptide due to flexibility, the core structure starting from Tyr41 could be determined and analysed, which superposes well with SSI-family proteins. ENZYMES: Chymotrypsin, EC3.4.21.1; griselysin (SGMPII, SgmA), EC3.4.24.27; snapalysin (ScNP), EC3.4.24.77; streptogrisin-A (SGPA), EC3.4.21.80; streptogrisin-B (SGPB), EC3.4.21.81; subtilisin BPN', EC3.4.21.62; transglutaminase, EC2.3.2.13; transglutaminase-activating metalloprotease (TAMP), EC3.4.-.-; tri-/tetrapeptidyl aminopeptidase, EC3.4.11.-; trypsin, EC3.4.21.4. DATABASES: The atomic coordinates and structure factors (PDB 6I0I) have been deposited in the Protein Data Bank (http://www.rcsb.org).