Complete inhibition of β-tryptase by tetramer dissociation and active site allostery due to a single antibody residue

Domain: neuroscience
Published: 2026-04-09
Editorial outcome: minor_revision

Abstract
Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of inflammatory responses in asthma, allergy and other diseases. Here we report an anti-β-tryptase antibody with a superior mechanism of action compared to others since it not only inhibits tetrameric β-tryptase, but also completely inhibits monomeric β-tryptase activity. The antibody binds to an exosite that causes tetramer dissociation as either an IgG or Fab and, in addition, allosterically alters the substrate binding cleft on monomers, thus preventing substrate binding and proteolysis. We solve the cryoEM structure of the complex, generate biochemical data and engineer point mutations to elucidate the allosteric path of inhibition. This ultimately reveals a single Asp to Gly mutation in CDR-L3 that only slightly impacts binding affinity, but completely eliminates inhibitory activity. Finally, we improve antibody inhibitory potency up to 4.7-fold by structure-based design creating new charge-charge interactions. This antibody may have enhanced efficacy and potential to assess the relevance of β-tryptase, including monomers, in biological and clinical settings.

Introduction
Mast cells play an important role in inflammation and host defense in response to foreign stimuli such as allergens, bacteria, viruses, and venoms and have also been implicated in a number of diseases including asthma, anaphylaxis, inflammatory bowel disease, and atherosclerosis1–3. IgE crosslinking on the surface of mast cells, through antigens or allergens, induces the release of secretory granules that contain histamine, heparin, cytokines, chemokines as well as high levels of various proteases1,4, in particular β-tryptase5. In the respiratory tract, this rapid secretion of active proteases has many physiological consequences such as increased collagen production by lung fibroblasts and proliferation and contraction of lung smooth muscle cells, specifically in type 2-low inflammation and type 2-low asthma6–10. Thus, potent and specific inhibitors of mast cell proteases may have broad therapeutic potential for diseases driven by excessive mast cell activation4,11, given that they do not impair their vital ‘normal’ physiological functions.

β-tryptase, a trypsin fold serine protease belonging to the S1A subfamily of S1 proteases (MEROPS peptidase database)12, is the most abundant protease stored in mast cell granules. Upon pro-peptide cleavage by cathepsin C13, β-tryptase forms tetramers in mast cell granules, which are stabilized by binding to serglycin proteoglycans carrying heparin glycosaminoglycan14. Extracellular β-tryptase monomers have often been considered to be inactive under physiological conditions15–18, but we and others have shown that monomeric tryptase can, in fact, retain activity in vitro at high heparin concentrations, since heparin also acts as a cofactor that increases activity18–22. The findings of the antibody we present herein provide a future opportunity to address the precise effect of heparin on monomeric tryptase activity in vivo, which is otherwise difficult to determine. Proteolytically active β-tryptase tetramers stimulate a range of inflammatory and tissue remodeling processes through cleavage of a variety of substrates including proteinase-activated receptor (PAR-2)7, vasoactive intestinal peptide (VIP)23, matrix metalloproteinases (MMPs)22,24, fibronectin, and collagen8, but in some cases, cleavage can also lead to inhibition of biological pathways25–27.

The crystal structures of active human β-tryptases show a frame-like, toroidal quaternary structure with a quasi 2-fold symmetry among the four individual protomers, surrounding a central pore with the four active sites facing toward the pore28,29. Positively charged surfaces outside the pore have been implicated in heparin binding that stabilizes the tetramer18,30. Due to the dimensions of the pore entrance (15×40 Å), access to the four active sites is limited to small peptide substrates and loops from larger proteins that are flexible enough to enter the pore. Common endogenous inhibitors of trypsin-like proteases such as alpha 1-antitrypsin, alpha 2-macroglobulin or Kunitz domain containing proteins are too large to access the active sites through the pore31,32. Only a small number of β-tryptase inhibitors in nature have been described, which include tick-derived protease inhibitor (TdPI)33, leech-derived tryptase inhibitor (LDTI)34,35, peptide leucine arginine (pLR)36 and cyclotheonamide E437. Engineered peptides derived from the cystine-knot peptides EETI-II38 and MCoTI-II39,40 as well as Bowman-Birk inhibitor peptides41 have been reported as potent β-tryptase inhibitors, but these molecules are not selective for β-tryptase and can block other trypsin-like serine proteases as well. Small molecule active site inhibitors of β-tryptase have also been described in the literature42–45 and some have shown therapeutic effects in guinea pig and sheep asthma models46,47 and in humans48, however, insufficient oral bioavailability, selectivity and/or tolerability have impeded further drug development efforts.

Inhibitory antibodies with drug-like properties and high specificity against their respective proteases can inhibit by either directly blocking the active site or indirectly modulating proteolytic activity via an allosteric mechanism49. We have previously described two inhibitory anti-β-tryptase antibodies with almost identical tryptase binding epitopes, that inhibit enzymatic activity allosterically by dissociating the active tetramer into monomers that are inactive at low heparin concentrations10,50. While these antibodies10,50 had promising activities in vitro and one was investigated in human clinical trials51, an antibody that completely inhibits both tetrameric and monomeric tryptase and can be used as a Fab or scFv may lead to the successful development of a therapeutic in the clinic. A lower MW inhibitor may increase the likelihood of reaching clinically relevant concentrations in the lower airway, a limiting factor in previous tryptase clinical trials51.

Here, we describe the mechanism of action (MOA) of an inhibitory anti-β-tryptase antibody (E82.AS). To the best of our knowledge, it binds to a previously unknown exosite, i.e., a binding site that is distinct from the ‘active site’, discovered by employing a series of biochemical, biophysical and structural biology experiments. We used hydrogen deuterium exchange mass spectrometry (HDX-MS) and cryogenic electron microscopy (cryoEM) to determine the β-tryptase:Fab interactions and discovered that E82.AS binding to an exosite changes the conformation of the active site cleft such that tryptase can no longer bind to substrates or active site inhibitors. Both the bivalent E82.AS IgG and the monovalent Fab dissociate tetrameric β-tryptase into completely inhibited monomers, even at high heparin concentrations. Based on structure-guided design, we introduced two mutations in the first complementarity-determining region (CDR) of the heavy chain (HC), CDR-H1, that improved the inhibitory potency of the antibody 4.7-fold.

Notably, we identified a single residue mutation—D93G in CDR-L3 of E82.AS—that has relatively little effect on β-tryptase binding, but no longer dissociates the tetramers into monomers nor has any inhibitory effect on enzymatic activity. This is a rare finding. Our high-resolution structure of the complex allowed us to trace structural changes stemming from the interaction of E82.AS D93 with the exosite on the β-tryptase surface and observe the allosteric path of tryptase inhibition into the active site in molecular detail. Insights into the path of allosteric inhibition may be applicable to inhibitor designs of other trypsin-like serine proteases, where a high degree of specificity and potency is required for therapeutic intervention.

Results
Rabbit immunization with recombinant β-tryptase and subsequent monoclonal antibody development by standard hybridoma technology resulted in a small number of specific anti-β-tryptase antibodies that also showed potent inhibition of tetrameric tryptase10. In this paper, tryptase or β-tryptase refers to the βI-tryptase subtype, which was used herein to generate all data. One of the antibody clones, E82.AS, a human chimeric IgG1 antibody, bound to β-tryptase with very high affinity, having a KD = 67 pM as measured by surface plasmon resonance (SPR) (Table 1). Both chimeric IgG and Fab E82.AS showed dose-dependent inhibition of tetrameric β-tryptase in a chromogenic enzyme activity assay containing 1 µg/mL heparin (Fig. 1a), having IC50 values of 47 nM and 86 nM, respectively. Epitope binning analysis of E82.AS using biolayer interferometry demonstrated that E82.AS did not compete with previously described inhibitory anti-βI-tryptase antibodies 31A.v1110 and E104.v250 (Supplementary Fig. 1). Both of these antibodies are structurally and mechanistically well understood, having very different MOAs, yet highly overlapping binding epitopes. This indicates that E82.AS binds to a distinct epitope on β-tryptase and interaction with this epitope is sufficient to inhibit enzymatic activity.

In order to determine whether the mechanism of β-tryptase inhibition by E82.AS is driven by tetramer dissociation into monomers, as found with 31A.v11 and E104.v2, we formed complexes of human β-tryptase tetramers with Fab E82.AS in the absence of heparin and analyzed them by size exclusion chromatography (SEC) and SDS-PAGE (Fig. 1b, c). β-tryptase tetramers mixed with excess Fab E82.AS eluted as a complex of both components with a retention volume of 14 mL (blue trace, Fig. 1b). This complex elutes with a larger volume than tetrameric tryptase alone (13 mL, gray trace Fig. 1b) and is indicative of tryptase monomers bound to Fab (lower molecular weight than tetramers), showing that Fab E82.AS binding to tetrameric β-tryptase dissociates tetramers into monomers.

We then tested whether Fab E82.AS causes β-tryptase tetramer dissociation via destabilization of the small interface or the large interface of the β-tryptase tetramer. To do this, we used previously described β-tryptase tetramer mutants Y75C and I99C (chymotrypsinogen numbering52 used throughout for β-tryptase), where the tetramers are covalently locked at their small (Y75C) or large (I99C) protomer interfaces by disulfide bonds and can only dissociate into dimers19. Complexes of these mutants bound to Fab E82.AS were analyzed by SEC and SDS-PAGE. Both tetramer mutants, Y75C and I99C, when bound to Fab E82.AS, formed complexes with retention volumes of 11.8 mL and 12.5 mL, respectively (orange and green traces, Fig. 1b), larger than the retention volume of 10.4 mL observed for tetrameric β-tryptase bound to 4 Fabs of E104.v150. This indicates that Fab E82.AS binding to tetrameric β-tryptase destabilizes both the small and the large protomer interfaces. Note that while E104.v1 and E104.v2 have identical CDR sequences and KD values of 0.5 nM for βI-tryptase monomers, they differ at two key Vernier residues (V71R and F78V), which resulted in destabilization of the small interface by E104.v2, but not by E104.v150.

We wondered whether the inhibition of β-tryptase catalytic activity by E82.AS was entirely attributable to its ability to dissociate tryptase tetramers. To test this, we compared E82.AS with another antibody, 31A.v11, which also dissociates tetramers, but neither alters the active site conformation of tryptase monomers10 when compared to tetramer28 (RMSD 0.53 Å) nor inhibits the residual enzymatic activity of the resulting tryptase monomers (Supplementary Fig. 2). Upon addition of excess active site inhibitor aprotinin (2 µM), this residual enzymatic activity from 31A.v11-bound tryptase was inhibited by 95.3% (Fig. 1d), still retaining some residual activity. Notably, when we added Fab E82.AS (1 µM) to 31A.v11-bound tryptase instead of aprotinin, we did not observe any detectable residual enzymatic activity (Fig. 1d). This suggests that in addition to tetramer dissociation, Fab E82.AS binding has an allosteric effect on the substrate binding site. To further examine this, we incubated a mixture of WT tetrameric β-tryptase and excess Fab E82.AS with the macromolecular active site inhibitor soybean trypsin inhibitor (STI). This resulted in a complex consisting of monomeric tryptase bound to Fab E82.AS, but which, importantly and unlike inhibitory antibody 31A.v1110, did not include STI, as shown by SEC and SDS PAGE analysis (red trace, Fig. 1b, c). Both experiments suggest that E82.AS-bound β-tryptase has an altered active site conformation, which is incompatible with binding of the macromolecular active site inhibitor STI binding or with proteolytic cleavage of a small chromogenic substrate.

Next, we sought to elucidate the Fab E82.AS binding epitope on WT β-tryptase. We conducted HDX-MS experiments with β-tryptase alone or in complex with Fab E82.AS. Amide protons in β-tryptase with slower exchange rates in the presence of E82.AS indicated that structural dynamics in these regions were affected upon Fab binding. We identified a number of peptides whose amide protons exchanged significantly slower compared to β-tryptase alone and calculated the individual protection factors for the respective amino acid positions. The highest positive protection factors (PF), which indicate slower HDX, clustered at residues I162 through A183, T186 through G196, and A213 through V223 (chymotrypsinogen numbering52) (ΔPF between 1 and 1.7). Many of these residues (180s loop and 220s loop) are part of the surface exposed activation domain loops that change conformation upon transition from zymogen to enzyme conformation. Furthermore, positive protections factors for residues D189, G193 and S195, which are part of the S1 specificity pocket and the oxyanion hole, both vital for catalysis, suggest structural changes in the active site. Residues 169-173d and 221-224 form a continuous surface near the large protomer interface, which is the most likely binding epitope of E82.AS (Supplementary Fig. 3). Consistent with the epitope binning results, this main cluster of residues does not overlap with the binding epitopes of 31A.v1110 or E104 (v1 or v2)50. All βI-tryptase residues with their respective calculated protection factors (log10 (ΔPF)) that represented slower or faster HDX in their amide bonds are listed in Supplementary Data 1.

While these experiments identified the likely epitope of E82.AS, the molecular details remained unclear as to how E82.AS dissociates tetramers into monomers and inhibits residual monomeric β-tryptase activity. This prompted us to employ cryoEM to gain high-resolution structural insights into the binding epitope of E82.AS as well as structural changes that occur in β-tryptase upon E82.AS binding. We formed a quaternary complex of WT β-tryptase, Fab E104.v2, Fab E82.AS with 3 mutations (K145E, E143S and Q199A in the kappa light chain) and an anti-kappa nanobody (Nb)53. The three mutations in the light chain (LC) of E82.AS abrogated anti-kappa Nb binding, ensuring that the Nb would only bind Fab E104.v2. Fab E104.v2 and the anti-kappa LC binding Nb were used as fiducial markers, increasing particle size and adding rigid features to aid in particle image alignment. This resulted in a structure at 3 Å global resolution with higher resolution at the Fab:β-tryptase interfaces (Fig. 2a, Supplementary Figs. 4 and 5, Supplementary Table 1). Consistent with the HDX-MS results, cryoEM data revealed that E82.AS binds to parts of the 170s loop, the preceding 160s helix and the 220s loop (Fig. 2b). The antibody paratope is characterized by contributions from the LC (49% buried surface area) and the HC (64% buried surface area) with overall buried surface area of 764 Å2 on β-tryptase. Antibody residues within 4 Å of β-tryptase are from CDR-L1 (V29, Y32), CDR-L3 (D92 to I95d), CDR-H2 (Y53, G54, S56), and CDR-H3 (F95, Y97, P98, A100) (Supplementary Fig. 6a). In β-tryptase, the epitope residues P132, G133, P161, M163, H166, I167, D169, A170, K171, H173, L173a, G173b, A173c, Y173d, N223, and R224 (chymotrypsinogen numbering52) are within 4 Å of E82.AS (Supplementary Fig. 6b). The same residues, except for P132 and G133 have ΔPF > 1, which is consistent with being part of the E82.AS binding epitope.

When we compared the Cα backbone of a β-tryptase protomer from the tetramer to the E82.AS-bound tryptase monomer, we discovered several significant conformational differences in the following areas: H36 to Y37b, G60 to D60e, R69 to Q81, Y173d to V173i, G184 to D194 and G216 to R224 (RMSD of the Cα backbone is 2.31 Å) (Fig. 2c). Furthermore, we cannot trace the Cα backbone in E82.AS-bound β-tryptase from I16 to K26 and W141 to K156 due to conformational heterogeneity (Fig. 2c). These findings have significant consequences for the active site and thus enzyme activity. Specifically, the oxyanion hole, which is critical for catalyzing proteolysis was non-existent due to a 4.6 Å shift of the main chain nitrogen atom of G193. Furthermore, the salt bridge interaction between D194 and the N-terminal primary amine of I16 was also non-existent, because the side chain of D194 was turned away by 90 degrees and thus the activation domain pocket was unavailable for stable N-terminal insertion (Fig. 2d). Finally, D189, which constitutes the important negative charge at the bottom of the S1 specificity pocket that interacts with positively charged P1 residues (Arg, Lys) of substrates, has moved away from the pocket; the distance between the two D189 Cβ atoms is 7.4 Å (Fig. 2e). Notably, catalytic triad residues H57, D102 and S195 were in similar positions (0.51 Å RMSD) compared to catalytically-competent β-tryptase. We provide a movie (Supplementary Movie 1) that allows a dynamic comparison of an ‘active’ β-tryptase protomer with a small molecule in the S1 pocket with an ‘inactive’ β-tryptase monomer bound to E82.AS. This movie shows the transition from the active enzyme conformation to a computational model of the ‘zymogen-like’ conformation upon E82.AS binding. The conformational changes for the N-terminus (I16), and residues D194, D189 and N223 are readily apparent.

Relative to the structure of tetrameric tryptase, we observed conformational changes from G60-D60e and R69-Q81 as well as structural heterogeneity from W141 to K156. These residues are at the small tetramer interface, which indicated increased dynamics in these regions of the protein compared to when they are part of the tetramer and have a defined structure for proper protomer interaction (Supplementary Fig. 7). It is difficult to conclude from this observation alone whether these conformational changes are directly induced by E82.AS binding or are due to the removal of stabilizing contacts to an adjacent monomer in the tetrameric state. Additionally, we observed conformational changes in β-tryptase at the E82.AS binding epitope near the large tetramer interface that could contribute to destabilization of this protomer-protomer interaction. D173g shifted by 5 Å and Y173d, which binds to a hydrophobic pocket in the neighboring tryptase protomer in the tetramer, rotated away from this pocket (Supplementary Fig. 8). The apparent destabilization of tetramers at both the large and small interfaces is a preferred MOA of E82.AS from the previously described inhibitory antibody E104.v2 that only destabilizes the small interface50.

Since S30 and Y31 from the HC of E82.AS were in close proximity to a negatively-charged patch on β-tryptase created by D169, D173g and D173h (Fig. 3a), we thought we might be able to engineer energetically favorable electrostatic interactions by introducing positively-charged amino acids at these positions. Indeed, HC.S30K.Y31K and HC.S30R.Y31R improved potency by 3.4-fold and 4.7-fold compared to WT, respectively (WT E82.AS IC50 = 64.2 nM; HC.S30K.Y31K IC50 = 18.8 nM; HC.S30R.Y31R E82.AS IC50 = 13.7 nM) (Fig. 3b), although we did not see an improvement in affinity to tryptase as measured by SPR (Table 1).

Next, we wanted to determine how E82.AS leads to tetramer dissociation and allosterically affects the active site. We inspected the binding epitope and noticed that D93 in CDR-L3 points directly toward the 220s loop of β-tryptase. In the E82.AS-bound structure, N223 is displaced by 4.5 Å relative to its position in the unbound tetrameric structure due to steric hindrance by the side chain of LC.D93 (Fig. 4a). To test whether this distortion of the 220s loop by LC.D93 leads to tetramer dissociation into monomers, we made a LC.D93G mutant, which eliminates the side chain, predicting that it would relieve the steric distortion of the 220s loop. Indeed, the E82.AS LC.D93G mutant still bound β-tryptase with very high affinity as determined by SPR (E82.AS LC.D93G KD = 788 pM) (Table 1). However, notably, this single point mutant no longer inhibited β-tryptase activity (Fig. 4b) nor dissociated β-tryptase tetramers into monomers as shown by SEC and SDS-PAGE (Fig. 5a, b). The mutations at HC.S30 and HC.Y31 described above that improved the inhibitory potency of WT E82.AS were not sufficient to rescue the lost inhibitory effect in the context of the LC.D93G mutant (Fig. 4c). The lack of tetramer dissociation with this Fab was also observed by negative stain EM (Fig. 5c), where the tetramer with 4 Fabs is clearly evident.

We hypothesized that the conformational changes in the 220s loop, instigated by the rearrangement of N223, were transduced to the active site via the disulfide bond between C191 and C220. This disulfide connects the 220s to the 180s loop and is displaced 5.2 Å at C191 and 2 Å at C220 in the E82.AS-bound structure (Fig. 4a) and thereby relays the structural rearrangement in the 220s loop to the 180s loop. To demonstrate this connection, we first stabilized the catalytically active conformation of the 180s loop (oxyanion hole and N-terminal insertion) in WT tetrameric tryptase with their reversible active site inhibitor Glu-Gly-Arg-chloromethylketone (EGR-cmk) covalently bound to S195 and then formed a complex with WT Fab E82.AS. SEC analysis showed a major peak in which four E82.AS Fabs were bound to active site-inhibited tetrameric tryptase with only a minor peak showing monomeric tryptase bound to E82.AS (Fig. 5a, b). This implies that the transduction of the structural changes in the 220s loop can be stopped if the active conformation in the 180s loop is stabilized and thus keeps the tetramer intact. Altogether, we conclude that E82.AS binding to tetrameric tryptase causes conformational changes in the 220s loop solely by E82.AS LC residue D93, which are further transduced via the disulfide bond C191-C220 to the 180s loop and subsequently to the active site. This leads to the release of I16 from the activation domain pocket, tetramer dissociation into monomers, and a transition to a zymogen-like conformation with no activity, even in the presence of high amounts of heparin.

Methods
Recombinant expression of WT and β-tryptase mutants
The sequence encoding mature wild-type (WT) human βI-tryptase (Uniprot Q15661) from I31 - P275 (I16 - P246 chymotrypsinogen numbering52) was cloned into a modified pAcGP67A vector behind the polyhedron promoter and the gp67 secretion signal sequence. Expression constructs contain an N-terminal His6-tag, followed by an enterokinase (EK) cleavage site directly fused to the mature N-terminal I16 of β-tryptase. Site-directed mutagenesis was performed using standard QuikChange protocols (Agilent, Santa Clara CA) to generate β-tryptase mutants. All constructs were confirmed by DNA sequencing. Recombinant baculovirus were generated using the Baculogold system (BD Biosciences, San Jose CA) in Sf9 insect cells (Expression Systems, Davis CA) following standard protocols. Tni insect cells (Expression Systems, Davis CA) were infected for large-scale protein production and harvested 48 h post-infection. The harvested media was supplemented with 1 mM NiCl2, 5 mM CaCl2 and 20 mM Tris pH 8, shaken for 30 min and then centrifuged for 20 min at 8500 x g to remove the cells and precipitate from media. The supernatant media was filtered through a 0.22 μm PES filter prior to loading onto a Ni-NTA affinity column.

Insect cell media containing secreted His6-tagged zymogen βI-tryptase (WT or mutant) was loaded onto a 10 mL Ni-NTA Superflow column (Qiagen, Germantown, MD) at a volumetric flow rate of 170 cm/h. The column was washed with 10 column volumes (CV) of wash buffer (20 mM Tris pH 8, 10 mM imidazole, 300 mM NaCl) and eluted with 8 CV elution buffer (20 mM Tris pH 8, 300 mM imidazole, 300 mM NaCl). Fractions assayed by SDS-PAGE containing βI-tryptase were pooled, concentrated and loaded onto an S200 column (GE Healthcare, Piscataway, NJ) for further purification by size-exclusion chromatography (SEC) using SEC buffer (10 mM MOPS pH 6.8, 2 M NaCl) at flow rates recommended by manufacturer. Fractions containing zymogen βI-tryptase monomer were pooled and concentrated.

Zymogen βI-tryptase was then cleaved overnight at room temperature at a concentration of 2 mg/mL in 10 mM MOPS pH 6.8, 0.2 M NaCl containing 0.5 mg/mL heparin (H3393; average MW~18 kDa; Sigma Aldrich, St. Louis, MO) and 0.1 mg/mL EK (NEB, Ipswich, MA). This step removes the N-terminal His6-tag and results in tetramerization and proteolytically active βI-tryptase, which has IVGG as the newly formed N-terminal sequence starting at residue 16. Tetrameric βI-tryptase was then subjected to SEC using an S200 column (GE Healthcare, Piscataway, NJ) in SEC buffer to purify tetrameric βI-tryptase by removing EK and any uncleaved zymogen βI-tryptase. βI-Tryptase mutants Y75C and I99C were purified by Ni-affinity chromatography as described above. Disulfide-linked βI-tryptase dimer mutants were then separated from non-disulfide linked βI-tryptase monomer mutants by SEC on an S200 column as above. Disulfide-linked dimer mutants were further processed by EK cleavage as described above for WT βI-tryptase to form active tetramers (mutants Y75C and I99C).

Generation and purification of anti-β-tryptase antibodies and the anti-kappa nanobody
Human chimeric anti-β-tryptase variants (rabbit variable light (VL) and variable heavy (VH)) were expressed by transient transfection of CHO K1 cells (WuXi Biologics). Antibodies were purified by affinity chromatography and SEC using standard methods (MabSelect SuRe; GE Healthcare, Piscataway, NJ). E82.AS is a human chimeric IgG1 antibody with the variable light (VL)-constant light (CL) rabbit disulfide changed to C80A and C171S. The anti-kappa nanobody53 was produced in HEK293 cells (ChemPartners) by transient transfection and purified using standard Ni-affinity chromatography followed by size exclusion chromatography using an S-75 column in 20 mM histidine acetate pH 5.5, 150 mM NaCl buffer.

Production of chimeric Fab E82.AS
Chimeric Fabs of the antibodies were prepared by incubating the antibody with lysyl endopeptidase (Wako Chemicals, Inc.) at 37 °C for 1 h at an enzyme to antibody ratio of 1:500. After 1 h, the reaction was quenched by adding 1/10 volume of 10% acetic acid and the Fab and Fc fragments were separated by cation exchange chromatography (50 mM Na-acetate buffer pH 5, NaCl gradient 0-1 M over 20 column volumes) using a 5 mL HiTrap SP HP column (GE Healthcare, Piscataway, NJ). The isolated Fabs were subjected to a final purification step on an S-200 SEC column (GE Healthcare, Piscataway, NJ) in PBS.

β-Tryptase enzymatic activity assay
The enzymatic activity of 1 nM tetrameric βI-tryptase was measured in 200 µL volume using 2 mM S-2288 chromogenic substrate (DiaPharma Group, Inc., West Chester, OH) was performed as previously described19 using TN (20 mM Tris pH 8.0, 150 mM NaCl), TNH1 (20 mM Tris pH 8.0, 150 mM NaCl, 1 µg/mL heparin) or TNH100 (20 mM Tris pH 8.0, 150 mM NaCl, 100 µg/mL heparin) buffers as indicated. The reaction velocity was determined by the rate at which p-nitroaniline (pNA) is released, which was measured spectrophotometrically at 405 nm using a SpectraMax M5e plate reader using Softmax Pro (v6.2.2) (Molecular Devices, Sunnyvale, CA). Inhibition plots were generated using 4-parameter fits from data collected from 3 independent experiments and statistical analysis was performed using KaleidaGraph (v5.0.6) (Synergy Software, Reading, PA).

Complex formation of βI-tryptase tetramers with Fabs
Tetrameric βI-tryptase, wild-type or mutants Y75C or I99C, were mixed with a 2-fold molar excess of Fab and incubated for 15 min at 25 °C. The mixture was then subjected to S200 GL size exclusion column (25 mL bed volume, GE Healthcare, Piscataway, NJ) chromatography in either tryptase SEC buffer (10 mM MOPS pH 6.8, 2 M NaCl) or TNH100 (20 mM Tris pH 8.0, 150 mM NaCl, 0.1 mg/mL Heparin) buffer. All fractions containing protein as indicated by absorbance at A280 nm were analyzed by SDS-PAGE under non-reducing conditions to identify the protein components in these fractions. SeeBlueTM Plus2 pre-stained protein standard (Thermo Fisher, Waltham, MA) was used for molecular weight markers for SDS-PAGE analysis.

Hydrogen deuterium exchange mass spectrometry analysis
Deuterium uptake rates of zymogen βI-tryptase monomer in the presence and absence of antibody E82.AS was measured to determine structural regions that are modified upon antibody binding. Bound samples contained 1:1 mixture of zymogen βI-tryptase and the respective antibody (98% bound using measured dissociation constants) prepared and incubated at 25 °C for 1 h. Zymogen βI-tryptase concentration prior to deuterium labeling was 30 μM in Ab bound and unbound samples. HDX-MS experiments consisted of diluting samples 20-fold into deuterium labeling buffer containing 20 mM histidine acetate at pD 7.0 (Serva Electrophoresis GmbH, Heidelberg, Germany) at 5 °C. Six labeling times (0.5, 2, 10, 54, 278, and 1440 min) were sampled in triplicate, quenched by lowering the pH to 2.5 and the addition of 2 M guanidinium chloride (Sigma Aldrich, St. Louis, MO) and 0.25 M TCEP (Sigma Aldrich, St. Louis, MO) using an extended, parallel arm LEAP HDX automation system (Trajan Scientific, Chronos software Version 5. 1. 10). Samples were first passed through an immobilized 50:50 Fungal protease/Pepsin column (NovaBioAssays) and loaded onto an online trap column (Acquity Vanguard C8, Waters) for desalting at 150 μL/min for 2 min at 0 °C. Peptide fragments were then separated by reversed-phased chromatography (35 μL/min) where they were introduced into the mass spectrometer by electrospray ionization (Thermo Q-Exactive, 120 kHz resolution at m/z 400) for mass analysis. To further minimize back-exchange, 0.04% v/v TFA was added to mobile phases containing 0.1% formic acid to adjust their pH approximately to 2.25. The levels of recovery on average are thus expected to be roughly 85%68.

Protein Metrics Byonic (Version 4.5.2) and Byologic (Version 4.5-53x64) software was used for peptide identification (FDR 2D filter of ≤ 0.0001) followed by ExMS 2.069 (Version 03 NOV 2018) to provide XIC’s and deuterium uptake levels for each peptide across the sampled time points using recommended settings was used to identify deuterated peptides and prepare extracted ion chromatograms, which were then analyzed for deuterium content using in-house implementations of an-population binomial analysis using a statistical F-test70. Prior to fitting isotopic distributions, degenerate charge states were combined by direct summation of equivalent peak intensities.

A total of 195 peptides (Supplementary Fig. 12) covering 88.8% of tryptase with an average redundancy of 10.60 unique peptides per residue were included for PF analysis using the empirical method71, with errors defined by the SEM of triplicate measurements. The value observed for the smallest peptide for each residue was taken as the change in protection, or ΔPF, for that residue. A spreadsheet, available in Supplementary Data 1 contains a coverage map, standard summary statistics72, βI-tryptase sequence in full length and chymotrypsinogen numbering, the empirical PF value along with the error in its measurement for all peptides used to define the PF plot, annotations for residues contacting the antibody E82.AS as well as those involved in allosteric changes that lead to inactivation of βI-tryptase upon binding.

To determine significance of the measured PF, we used our previously published combinatorial approach to estimate error50. This estimation gives an asymmetric error about the mean, thus the term positive and negative below. A value was considered significant if its magnitude exceeded the SEM. For protected regions (log(PF) > 0), the value had to be larger than the negative error. For deprotected regions (log(PF) < 0), the absolute value had to be greater than the positive error. Nonsignificant values are reported as zero in the Smallest Peptide tab of Supplementary Data 1. In regions where the PF was obviously much larger than could be estimated by this empirical method, the peptide was flagged as stated in the Notes column. Finally, the sequence and numbering of the protein used for HX experiments as well as chymotrypsinogen numbering52 is included for any necessary disambiguation as a tab in this spreadsheet.

Cryogenic electron microscopy data collection and image processing
The E82.AS:βI-tryptase:E104.v2:Nb complex was formed by incubating all components at a 1:1:1:1 ratio for 1 hour before running through an S200 gel filtration column in 20 mM HEPES pH 7.5, 100 mM NaCl buffer. The peak fraction was diluted to 0.25-0.3 mg/mL in 20 mM HEPES pH 7.5, 100 mM NaCl directly before vitrification, and 2.5 μL sample was applied to glow-discharged holey gold 0.6/1 300-mesh grids (Quantifoil). Grids were blotted for 3 s at 7 force before being plunge vitrified in liquid ethane using a Mark IV Vitrobot (Thermo Fisher). The blotting chamber was maintained at 4 °C and 100% humidity during freezing.

Movies were collected using a Titan Krios G3i (Thermo Fisher) outfitted with a K3 camera and Bioquantum energy filter (Gatan). The K3 detector was operated in non-CDS counting mode and the energy filter slit width was set to 20 eV. Movies were collected at a nominal magnification of 105,000×, physical pixel size 0.838 Å pixel−1, with a 50 μm C2 aperture and 100 μm objective aperture at a dose rate of 15 e− pixel−1 s−1. A total dose of 64 e− Å−2 was collected as a 59-frame movie, resulting in a 3 s movie with 1.08 e− per frame. Data were collected using semi-automated imaging scripts in SerialEM (v4.1.25)73. 4,710 movies were collected in a 3×3 image shift pattern at 0° tilt, 2739 at 15°, 3210 at 30°, and 2442 at 40° in consecutive data collections. Beam tilt was corrected for using SerialEM’s image shift vs coma calibration. Raw cryo-EM movies have been deposited in EMPIAR74 as EMPIAR-13094.

Data were processed on-the-fly through 2D classification using cryoSPARC (v4.2.1 and v5.1.0)75. Movies were motion corrected using patch motion correction, CTF information was estimated using patch CTF estimation, and micrographs were curated based off of CTF resolution fit. Templates created from a ~9 Å map from data collected on a Glacios (Thermo Fisher) outfitted with a K2 camera (Gatan) were used for particle picking. Template picking yielded 8,899,017 picks, roughly evenly split across the collections. These were extracted in a 420-pixel box, and binned to 128 pixels. 2D classification was run with default settings and a circular mask of 140 Å. ‘Good’ (showing at least one Fab) class averages were selected and 6,338,581 particles were moved into 3D sorting. Four ab initio classes were generated from the 0° tilt data, 2,262,677 particles, one showing density for a single Fab, one resembling an amorphous blob, one showing the expected complex of tryptase and two Fabs, and one showing roughly two copies of tryptase with two Fabs. All particles were subjected to four rounds of heterogeneous refinement using these volumes as starting models, with two copies of the ‘correct’ complex. 1,160,676 particles were re-extracted in a 450-pixel box, and binned to 256 pixels. One round of heterogeneous refinement was run with no downsampling. 716,327 particles were re-extracted in a 450-pixel box, and binned to 324 pixels, giving a final pixel size of 1.164 Å pixel−1. One round of heterogeneous refinement was run, particles downsampled to 256 pixels. Two classes, constituting 680,181 particles, were subjected to non-uniform refinement. Both classes clearly showed the intact complex, with one centered on Fab E82.AS and one centered on tryptase. The classes with tryptase centered was used as an initial model and filtered to 15 Å before refinement. Non-uniform refinement was run after reference-based motion correction, per-particle defocus, per-group CTF parameter optimization, and duplicates were removed. The final map from 672,348 particles was estimated to be 3 Å by gold-standard FSC. Local resolution, orientation diagnostics and local filtering jobs were run on the final map.

For β-tryptase and E104.v2, a previous crystal structure (PDB: 6VVU) was used as an initial model. An Alphafold2 model of Fab E82.AS was used as an initial model. The nanobody was rigid fit into the density from the crystal structure PDB: 6ANA. Because of the low resolution of the map in this region, side chains were not assigned. The initial templates were rigid fit into the cryoEM map of E82.AS:βI-tryptase:E104.v2:Nb using ChimeraX (v1.7.1)76. The models were manually fit and mutated in E82.AS and E104.v2 with ISOLDE (v1.6)77. The model was refined in Phenix real space refinement78. Model geometry and map-to-model statistics were assessed using the EM validation check in Phenix (v1.20.1-4487). CryoEM data collection, refinement and validation statistics are presented in Supplementary Table 1. Figures were prepared in ChimeraX and PyMol (version 3.1.3)79.

To create a complete structure to enable a morph between the enzyme and zymogen conformations, otherwise disordered loop residues (residues 16-25 and residues 141-155) were grafted into our E82.AS/E104.v2-bound structure PDB: 9MNB from a β-tryptase protomer of 1A0L. The peptide geometry of the fusion points was improved using Coot’s Bond regularization tools. The overall positions of these two loops were then manually adjusted outwards using the ‘sculpting’ function of PyMOL to create a potential zymogen-like conformational model for these specific areas while maintaining reasonable backbone geometry. Supplementary Movie 1 was made in ChimeraX using the morph function between the active enzyme conformation of β-tryptase as seen in the tetrameric state (PDB: 1A0L) and this computationally-derived fusion that represents the E82.AS/E104.v2-bound β-tryptase structure in its inhibited zymogen-like structure.

Epitope binning and competition binding of antibodies E82.AS, 31A.v11 and E104.v2
The epitope binning assay, a label free dip and read assay, was run using Octet RED384 system (Sartorius Corporation, USA). The assay buffer was Phosphate-Buffered Saline (PBS) containing 0.5% Bovine Serum Albumin and 0.05% Polysorbate 20. Briefly biotinylated human β-tryptase, diluted at 10 μg/mL in assay buffer, was captured using streptavidin Octet biosensor tip (cat# 18-5019, Sartorius Corporation, USA) for 200 s followed by binding of the first mAb, E82.AS hIgG1 at 20 μg/mL for 600 seconds to saturation. The cross-blocking was observed by dipping the saturated sensor into the second mAb at 5 μg/mL for 300 s. An increase in signal upon dipping into the second mAb indicates that both antibodies can bind simultaneously to β-tryptase and bind distinct epitopes.

Affinity and binding kinetics measurements of E82.AS variants by surface plasmon resonance
Antibody binding affinity was determined using a capture assay using the Biacore T200 system from Cytiva, USA. Briefly anti-human capture antibody was covalently immobilized onto a CM5 chip. Subsequently, the ligand, anti-β-tryptase antibodies with a human backbone, were captured on the chip for 20 s. This was followed by passing different concentrations of the analyte, zymogen βI-tryptase monomer, over the chip. The resulting binding kinetic data was then collected and analyzed using Cytiva evaluation software, employing a 1:1 binding model.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Review report.\nThe manuscript by Henry R Maun et al is a very interest study of antibodies against the human beta-tryptase that modifies\nthe region close to the active site and also dissociates the tryptase tetramers, which result in complete inhibition of the\nenzyme activity.\nAccording to the authors the aim of the study is to obtain antibodies to block the tryptase activity for therapeutic use.\nThe tryptase locus has been conserved in its overall structure during more than 180 million years of mammalian evolution\nfrom marsupials to humans (1). A tryptic enzyme is also present in the mast cell like cells in a tunicate, an early cordate,\nindicating that tryptic enzymes are an important component of the mast cell and its function in immunity (2). I therefore think\nthe authors should modify their quite single-minded view that the tryptase is only negative and should be removed by\ntherapeutic antibodies. I think the manuscript needs a more balanced view of the mast cell enzymes as if they were only\nnegative would have been lost during evolution a long time ago. The human beta tryptase have together with the human\nmast cell chymase been shown to cleave a very restricted number of human cytokines and chemokines indicating a potent\nrole of these mast cell enzymes in controlling excessive TH2 immunity (3) Removing the tryptase by antibody targeting\nwould potentially risk a deteriorating allergic response by not removing excessive TH2 cytokines by the mast cell enzymes.\nThis effect would most likely not be seen during short term use but after prolonged use if such an antibody. I think the authors\nneed to modify their background information also to include potential negative effects by removing these enzymes.\nThe use of a chimeric antibody where the V region is of Rabbit origin will also result in antibody formation and a severe\nreduction in efficacy within a few weeks. A transfer of the CDR regions into a fully human V region background would\ntherefore be needed before testing the antibody for human use. This is something that needs to be discussed in the\nmanuscript to highlight the strict need for optimal humanized antibodies for clinical use.\nThe study of the structural changes induced by the antibody binding is however highly interesting for the function of the\nenzyme. For us non-structural biologists, a more easily interpreted illustration showing the structural changes of the region of\nthe active site and the oxyanion hole affecting the cleavage efficacy would be fantastic to make the manuscript more easily\nviewed by non-structural biologists, if possible. Is the position of the amino acids of the surrounding the active site after\nantibody binding similar to the tryptase having the N-terminal activation peptide attached? Then the enzyme is totally\ninactive similar to the situation with the antibody bound.\nProduction of an antibody against the mouse counterpart to the human beta-tryptase the mMCP-6 would potentially be a\nvery interesting tool. This to in mice test the potential beneficial or negative effects by reducing the mast cell tryptase levels\nby antibody treatment, studies that have to be performed before testing in clinical tests in humans. Something that could be\ndiscussed in a revised manuscript.\nReferences\n1. Reimer JM, Samollow PB, Hellman L. High degree of conservation of the multigene tryptase locus over the past 150-200\nmillion years of mammalian evolution. Immunogenetics. 2010;62(6):369-82.\n2. Wong GW, Zhuo L, Kimata K, Lam BK, Satoh N, Stevens RL. Ancient origin of mast cells. Biochem Biophys Res\nCommun. 2014;451(2):314-8.\n3. Fu Z, Akula S, Thorpe M, Hellman L. Highly Selective Cleavage of TH2-Promoting Cytokines by the Human and the\nMouse Mast Cell Tryptases, Indicating a Potent Negative Feedback Loop on TH2 Immunity. International journal of\nmolecular sciences. 2019;20(20).\nLars Hellman PhD Professor\nUppsala University\nDept. Of Cell and Molecular Biology\nBMC, Box 596\nSE-751 24 Uppsala\nSWEDEN", "reviewer_2": "This study by Maun et al. characterizes a novel inhibitory antibody that targets human β-tryptase—a trypsin-like protease\nimplicated in inflammatory diseases such as asthma and anaphylaxis. The antibody demonstrates an exceptional\nmechanism of action: by binding to a novel exosite it not only dissociates β-tryptase tetramers into inactive monomers but,\nquite interestingly, also allosterically blocks monomeric enzymatic activity by altering the substrate binding cleft.\nUsing cryo-EM and HDX-MS and other biochemical methods, the authors identified a single residue (D93) in the antibody's\nlight chain critical for allosteric inhibition, revealing the structural pathway from antibody binding to active site perturbation.\nMutating this residue (D93G) eliminated inhibitory function without affecting binding, a rare and interesting finding.\nOverall, I find the paper interesting, and it offers a detailed molecular explanation of antibody-mediated allosteric inhibition\nand could open up for developing new therapeutics against mast cell-related diseases. Judging my primary area of\nexpertise, the HDX-MS experiments have been well conducted and the conclusions drawn are sound. I am positive towards\npublication, subject to addressing my comments below.\nComments:\nThe authors should follow the HDX-MS community guidelines (Masson et al. 2019) and include HDX Summary and HDX\nData tables so that the data is more accessible.\nThe authors should include examples (in Supporting info) of uptake curves where they see the largest differences in HDX –\nboth from the epitope (180s and 220s loops for instance) – as well as some for the allosteric effects in the active site. The\nmagnitudes of these differences are important to show in my opinion. On that note, the authors should describe in more\ndetail how they decide what effects are significant and their statistical threshold etc.\nThe authors are also encouraged to show a peptide map showing which peptides exhibit statistically significant differences\n– that information is difficult for the reader to discern from Supporting Fig.S2 etc.", "reviewer_3": "Maun et al present the biophysical, biochemical, and structural characterization of an antibody that inhibits tryptase by\ndestabilizing the active tetramer and trapping the monomer in an inactive conformation by virtue of an allosteric interaction at\nan exosite. They demonstrate the validity of their findings by mutations that improve binding or else abolish the allosteric\ninhibitory mechanism and develop a structure-based model for the allosteric pathway. Overall, the manuscript is clearly\nwritten. The cryo-EM map, and methods are of high quality and the HDX experiments strengthen the interaction analysis.\nRelatedly, the detailed methods for cryo-EM data collection and processing workflow are valuable and highlight the lengths\ntaken to solve the structure. This manuscript is well-suited for Nature Communications as it includes findings directly\napplicable to human therapies and protease aficionados. I applaud the authors on this exciting study, which should be\naccepted.\nI have only minor points to polish the manuscript and strengthen its value to the community:\n1) Given the broad audience of Nature Communications, the authors could define terms like “exosite” and generally give the\nmanuscript an edit to ensure other terms or concept specific of the field are clearly described.\n2) Figure 1B/C – It would strengthen the panels to include a positive control of STI co-migrating with Tryptase in this\nexperiment.\n3) There is an epitope binning BLI experiment confirming the E82.AS epitope does not overlap with 31A.v1110 and E104.v2\nmentioned as “data not shown” – would the authors consider adding this data the the supplement?\n4) Would the authors please check if this sentence in the discussion, “Increasing potency of E82.AS, by introducing the\ndouble mutant HC.S30R.Y31R, did not rescue the loss of inhibitory activity created by the LC.D93G mutation, indicating that\nLC.D93 is the key driver of the allosteric inhibition (Fig. 4b)” is referring to Sup Fig 8? In any case, I suggest moving S8 to the\nmain figures.\n5) The authors should add the atomic model for the nanobody to the pdb. It has reasonable density, and based it being\nreferenced, I believe its sequence is published. The stated rationale for its omission is that it “was used solely as a fiducial\nmarker for data processing” which would also apply to the Fab E104.v2 that was included in the model.\n6) If possible, I recommend the authors deposit the raw cryo-EM movies to EMPIAR.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Author Reply to Reviewer Comments\nReviewer #1 (Remarks to the Author):\nThe manuscript by Henry R Maun et al is a very interest study of antibodies against the human\nbeta-tryptase that modifies the region close to the active site and also dissociates the tryptase\ntetramers, which result in complete inhibition of the enzyme activity.\nAccording to the authors the aim of the study is to obtain antibodies to block the tryptase\nactivity for therapeutic use.\nThe tryptase locus has been conserved in its overall structure during more than 180 million\nyears of mammalian evolution from marsupials to humans (1). A tryptic enzyme is also present\nin the mast cell like cells in a tunicate, an early cordate, indicating that tryptic enzymes are an\nimportant component of the mast cell and its function in immunity (2). I therefore think the\nauthors should modify their quite single-minded view that the tryptase is only negative and\nshould be removed by therapeutic antibodies. I think the manuscript needs a more balanced\nview of the mast cell enzymes as if they were only negative would have been lost during\nevolution a long time ago. The human beta tryptase have together with the human mast cell\nchymase been shown to cleave a very restricted number of human cytokines and chemokines\nindicating a potent role of these mast cell enzymes in controlling excessive TH2 immunity (3)\nRemoving the tryptase by antibody targeting would potentially risk a deteriorating allergic\nresponse by not removing excessive TH2 cytokines by the mast cell enzymes. This effect would\nmost likely not be seen during short term use but after prolonged use if such an antibody. I\nthink the authors need to modify their background information also to include potential\nnegative effects by removing these enzymes.\nReferences\n1. Reimer JM, Samollow PB, Hellman L. High degree of conservation of the multigene tryptase\nlocus over the past 150-200 million years of mammalian evolution. Immunogenetics.\n2010;62(6):369-82.\n2. Wong GW, Zhuo L, Kimata K, Lam BK, Satoh N, Stevens RL. Ancient origin of mast cells.\nBiochem Biophys Res Commun. 2014;451(2):314-8.\n3. Fu Z, Akula S, Thorpe M, Hellman L. Highly Selective Cleavage of TH2-Promoting Cytokines by\nthe Human and the Mouse Mast Cell Tryptases, Indicating a Potent Negative Feedback Loop on\nTH2 Immunity. International journal of molecular sciences. 2019;20(20).\nAuthors: We thank the reviewer for their biological perspective on our manuscript. We fully\nrecognize that tryptase plays a very important role in mast cell biology. We certainly do not\nconsider tryptase to be a ‘bad’ enzyme and are surprised that this reviewer infers that it should\n1\nbe ‘removed’ – that is absolutely not what we intended to say. Any regulation of tryptase, like all\nproteases, is all about balance – too much or too little proteolysis of its substrates can lead to a\nphysiological imbalance that could have adverse consequences. The purpose of developing an\ninhibitor for any clinical condition is definitely not to ‘remove’ the protease and its function,\nrather to help to regulate it such a way as to achieve ‘normal’ function.\nTo address the concerns of the reviewer, we have added the following text to highlight the\nnormal ‘positive’ role of tryptase in a more balanced manner to explicitly point out its very\nimportant normal physiological functions. We have added a key new reference (Ref 66) to the\npaper as suggested by the reviewer. 66. Fu, Z., Akula, S., Thorpe, M. & Hellman, L. Highly\nselective cleavage of TH2-promoting cytokines by the human and the mouse mast cell tryptases,\nindicating a potent negative feedback loop on TH2 immunity. Int J Mol Sci 20, 5147 (2019).\nWhile this reviewer points out the conserved evolutionary aspects of the tryptase locus, we do\nnot think additional text or references are needed to convey the physiological importance of\ntryptase.\nNew Text (Introduction) highlighted in blue; original text in black: Thus, potent and specific\ninhibitors of mast cell proteases may have broad therapeutic potential for diseases driven by\nexcessive mast cell activation4, 11, given that they do not impair their vital ‘normal’ physiological\nfunctions.\nNew text (Discussion): “Any inhibition of tryptase in a clinical setting must be taken with great\ncare. While excess tryptase may lead to adverse physiological effects2,4,6, tryptase also has\nimportant vital roles as well. Mast cell tryptase cleaves a very restricted number of human\ncytokines (TSLP, IL-21) and chemokines (MCP3, MIP-3b, and eotaxin), implying an important\nimmune regulatory role in controlling excessive TH2-mediated inflammation66. Prolonged\ninhibition might lead to a diminished allergic response as well as excessive levels of TH2\ncytokines. Thus the extent to which tryptase inhibition may have clinical benefit must be\nbalanced with its inherent positive functions.” We added new ref 66 to the references and\nrenumber other references accordingly.\nThe use of a chimeric antibody where the V region is of Rabbit origin will also result in antibody\nformation and a severe reduction in efficacy within a few weeks. A transfer of the CDR regions\ninto a fully human V region background would therefore be needed before testing the antibody\nfor human use. This is something that needs to be discussed in the manuscript to highlight the\nstrict need for optimal humanized antibodies for clinical use.\nhighly preferable over their chimeric counterparts as clinical agents. We have added the\nfollowing paragraph to the discussion section address this concern.\nNew text: “While E82.AS is a potent inhibitor of human β-tryptase, it still contains rabbit\nvariable domain sequences. Prior to testing this antibody in the clinic, full humanization would\nbe required for developing it as a therapeutic to avoid any unwanted immunogenicity. This\nprocess would involve grafting the CDRs in a human germline as we have previously described50.\nThe study of the structural changes induced by the antibody binding is however highly\ninteresting for the function of the enzyme. For us non-structural biologists, a more easily\ninterpreted illustration showing the structural changes of the region of the active site and the\noxyanion hole affecting the cleavage efficacy would be fantastic to make the manuscript more\neasily viewed by non-structural biologists, if possible. Is the position of the amino acids of the\nsurrounding the active site after antibody binding similar to the tryptase having the N-terminal\nactivation peptide attached? Then the enzyme is totally inactive similar to the situation with the\nantibody bound.\nAuthors: We appreciate the challenge that non-structural biologists may have with figures\ndepicting the molecular details of the structure. We believe the figures submitted provide a very\nclear picture of what takes place upon binding of this antibody. There are no structures of\ntryptase having the N-terminal activation peptide attached (i.e. zymogen tryptase). To help this\nreviewer and others better appreciate and understand the structural details, we now include\nthe following text and a Supplementary Movie. We hope this will be helpful to readers to\nprovide a better visual understanding of the conformational changes that take place.\nNew Text: “We provide a Supplementary Movie that allows a dynamic comparison of an ‘active’\nβ-tryptase protomer with a small molecule in the S1 pocket with an ‘inactive’ β-tryptase\nmonomer bound to E82.AS. This movie shows the transition from the active enzyme\nconformation to a computational model of the ‘zymogen-like’ conformation upon E82.AS\nbinding. The conformational changes for the N-terminus (I16), and residues D194, D189 and\nN223 are readily apparent.”\nNew Methods for the Supplementary Movie: “To create a complete structure to enable a\nmorph between the enzyme and zymogen conformations, otherwise disordered loop residues\n(residues 16-25 and residues 141-155) were grafted into our E82.AS/E104.v2-bound structure\n(PDB:9MNB) from a β-tryptase protomer of 1A0L. The peptide geometry of the fusion points\nwas improved using Coot's Bond regularization tools. The overall positions of these two loops\nwere then manually adjusted outwards using the 'sculpting' function of PyMOL to create a\n3\npotential zymogen-like conformational model for these specific areas while maintaining\nreasonable backbone geometry. The Supplementary Movie was made in ChimeraX using the\nmorph function between the active enzyme conformation of β-tryptase as seen in the\ntetrameric state (PDB:1A0L) and this computationally-derived fusion that represents\nthe E82.AS/E104.v2-bound β-tryptase structure in its inhibited zymogen-like structure.”\nNew Supplementary Movie legend. “The movie shows the transition from the active enzyme\nconformation as found in 1A0L to a model of a ‘zymogen-like’ conformation\nupon E82.AS binding. Unmodeled loops in the E82.AS structure were artificially built in to create\na potential zymogen-like conformational model for these specific areas while maintaining\nreasonable backbone geometry. The active conformation contains a small molecule inhibitor\n(amido phenyl pyruvic acid (green)) bound in the S1 pocket. Catalytic triad residues H57, D102\nand S195 (cyan) and side chain residues for D194, D189 and N223 (beige) are shown in\nsticks. E82.AS light chain residue D93 (light blue) is shown in sticks.”\nProduction of an antibody against the mouse counterpart to the human beta-tryptase the\nmMCP-6 would potentially be a very interesting tool. This to in mice test the potential beneficial\nor negative effects by reducing the mast cell tryptase levels by antibody treatment, studies that\nhave to be performed before testing in clinical tests in humans. Something that could be\ndiscussed in a revised manuscript.\nAuthors: We agree that an antibody against mMCP-6, the murine counterpart of human β-\ntryptase would be an interesting tool to have, although the best animal models for asthma are\nin other species and therefore would not really help us advance that biology and physiology\ntowards clinical efforts. Based on our experience, the implied suggestion to have a mMCP-6\nspecific inhibitor is well beyond the scope of this work and would require a very significant\neffort. While it would be nice to have such an antibody, there is no guarantee that an antibody\nwith the precise characteristics of E82.AS could even be generated. Of the 16 residues on\nhuman β-tryptase that are within 4 Å of E82.AS, 7 of those are different in murine MCP-6, which\nlikely explains the specificity for human β-tryptase.\nReviewer #2 (Remarks to the Author):\nThis study by Maun et al. characterizes a novel inhibitory antibody that targets human β-\ntryptase—a trypsin-like protease implicated in inflammatory diseases such as asthma and\nanaphylaxis. The antibody demonstrates an exceptional mechanism of action: by binding to a\n4\nnovel exosite it not only dissociates β-tryptase tetramers into inactive monomers but, quite\ninterestingly, also allosterically blocks monomeric enzymatic activity by altering the substrate\nbinding cleft.\nUsing cryo-EM and HDX-MS and other biochemical methods, the authors identified a single\nresidue (D93) in the antibody's light chain critical for allosteric inhibition, revealing the\nstructural pathway from antibody binding to active site perturbation. Mutating this residue\n(D93G) eliminated inhibitory function without affecting binding, a rare and interesting finding.\nOverall, I find the paper interesting, and it offers a detailed molecular explanation of antibody-\nmediated allosteric inhibition and could open up for developing new therapeutics against mast\ncell-related diseases. Judging my primary area of expertise, the HDX-MS experiments have been\nwell conducted and the conclusions drawn are sound. I am positive towards publication, subject\nto addressing my comments below.\nAuthors: The authors thank this reviewer for their kind words and the thorough review of our\nwork. Please find point-by-point responses to all of their comments below.\nComments:\nThe authors should follow the HDX-MS community guidelines (Masson et al. 2019) and include\nHDX Summary and HDX Data tables so that the data is more accessible.\nAuthors: This information was present previously but not directly mentioned in the main text.\nWe have now called additional attention to it by stating directly that standard summary\nstatistics are available in Supplementary File SI_HXPFexcel.xlsx. See the new text below.\nThe authors should include examples (in Supporting info) of uptake curves where they see the\nlargest differences in HDX – both from the epitope (180s and 220s loops for instance) – as well\nas some for the allosteric effects in the active site. The magnitudes of these differences are\nimportant to show in my opinion.\nAuthors: We thank the reviewer for this suggestion. We recognize that much information can be\nlost through translating magnitudes onto structures with color; indeed, this is important\ninformation and to assist the reader, we have now annotated important regions in the revised\nSupplementary File SI_HXPFexcel.xlsx. Namely, we have highlighted residues that are within 4 Å\nof antibody E82.AS as seen in the cryo-EM structure, key residues that are in the active site and\nnot in contact with E82.AS, catalytic triad residues, and the disulfide bond that connects 220s\nand 180s loop have all been annotated. See the new text below.\nOn that note, the authors should describe in more detail how they decide what effects are\nsignificant and their statistical threshold etc.\nempirical PF in the main text and pasted it below for convenience. (Please note that the black\ntext was present in the submitted manuscript and the grey text has been deleted)\nOriginal text: A spreadsheet, available in extended data (Supplementary File SI_HXPFexcel.xlsx)\ncontains the values of each peptide used in this way, along with annotations for regions where\nthe ΔPF exceeded our ability to quantify using the empirical method and therefore a value was\nimputed (usually either log10(ΔPF) =1 or 2, see notes in Supplementary File SI_HXPFexcel.xlsx).\nNew text: “A spreadsheet, available in extended data (Supplementary File SI_HXPFexcel.xlsx)\ncontains a coverage map, standard summary statistics72, the empirical PF value along with the\nerror in its measurement for all peptides measured, along with a tab that annotates residues\ncontacting antibody E82.AS, as well as allosteric changes that lead to inactivation of βI-tryptase\nupon binding.”\n“To determine significance of the measured PF, we used our previously published combinatorial\napproach to estimate error50. This estimation gives an asymmetric error about the mean, thus\nthe term “positive” and “negative” below. A value was considered significant if its magnitude\nexceeded the SEM. For protected regions (log(PF)>0), the value had to be larger than the\nnegative error. For deprotected regions (log(PF)<0), the absolute value had to be greater than\nthe positive error. Nonsignificant values are reported as zero in the Smallest Peptide tab of\nSupplementary File SI_HXPFexcel.xlsx. In regions where the PF was obviously much larger than\ncould be estimated by this empirical method, the peptide was flagged as stated in the Notes\ncolumn.”\nWe have added the following citation (new Ref 72) and renumbered other citations as\nappropriate: 72. Masson, G. R. et al. Recommendations for performing, interpreting and\nreporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Nat\nMethods 16, 595-602 (2019).\nThe authors are also encouraged to show a peptide map showing which peptides exhibit\nstatistically significant differences – that information is difficult for the reader to discern from\nSupporting Fig.S2 etc.\nAuthors: We agree with the reviewer. In fact, all of this information was present in the\nSupplementary File SI_HXPFexcel.xlsx submitted with the manuscript. Only significant changes\nin protection factors are listed in the Smallest Peptide tab of this spreadsheet. Those that are\ndeemed non-significant are set to a value of zero. PFs along with errors for all peptides are\nshown.\naberration in one of the columns and have now corrected that in the revised file.\nReviewer #3 (Remarks to the Author):\nMaun et al present the biophysical, biochemical, and structural characterization of an antibody\nthat inhibits tryptase by destabilizing the active tetramer and trapping the monomer in an\ninactive conformation by virtue of an allosteric interaction at an exosite. They demonstrate the\nvalidity of their findings by mutations that improve binding or else abolish the allosteric\ninhibitory mechanism and develop a structure-based model for the allosteric pathway. Overall,\nthe manuscript is clearly written. The cryo-EM map, and methods are of high quality and the\nHDX experiments strengthen the interaction analysis. Relatedly, the detailed methods for cryo-\nEM data collection and processing workflow are valuable and highlight the lengths taken to\nsolve the structure. This manuscript is well-suited for Nature Communications as it includes\nfindings directly applicable to human therapies and protease aficionados. I applaud the authors\non this exciting study, which should be accepted.\nAuthors: We thank this reviewer for these very positive comments in their assessment of our\nwork.\nI have only minor points to polish the manuscript and strengthen its value to the community:\n1) Given the broad audience of Nature Communications, the authors could define terms like\n“exosite” and generally give the manuscript an edit to ensure other terms or concept specific of\nthe field are clearly described.\nAuthors: We thank the reviewer for this suggestion. We recognize that the term exosite may not\nbe widely used or understood by those in other fields. The term ‘exosite(s)’ generally refers to\nputative binding site(s) on the surface of the protease that differs from the ‘active site’. For\nserine proteases, the active site refers to the area comprising the Asp102-His57-Ser195 catalytic\ntriad as well as the S1 and S1’ pockets for substrate binding (see Schechter I, Berger A. On the\nsize of the active site in proteases. I. Papain. Biochem Biophys Res Commun. 1967 Apr\n20;27(2):157-62). The substrate cleft – typically from S5 to S5’ that binds substrates from P5 to\nP5’ – is generally not considered an exosite. Binding of proteins or other ligands at an exosite\nmay (in the case of inhibitors) allosterically modify the active site conformation such that it no\nlonger catalyzes substrate hydrolysis. Proteins (e.g. cofactors) or other ligands that bind at\nexosites may also allosterically activate proteases to a more active conformation.\nintroduction to briefly explain that an exosite is simply a binding site that is distinct from the\n‘active site’. We think this is sufficient for readers.\nOriginal text: Here we describe the mechanism of action (MOA) of a novel inhibitory anti--\ntryptase antibody (E82.AS) employing a series of biochemical, biophysical and structural biology\nexperiments.\nNew text: “Here we describe the mechanism of action (MOA) of a novel inhibitory anti--\ntryptase antibody (E82.AS) that binds to a novel exosite, i.e. a binding site that is distinct from\nthe ‘active site’, employing a series of biochemical, biophysical and structural biology\nexperiments.”\n2) Figure 1B/C – It would strengthen the panels to include a positive control of STI co-migrating\nwith Tryptase in this experiment.\nAuthors: We thank the reviewer for this suggestion. This experiment has been carried out\npreviously and reported in Maun HR, et al. An Allosteric Anti-tryptase Antibody for the\nTreatment of Mast Cell-Mediated Severe Asthma. Cell 179, 417-431 e419 (2019). This paper also\nshows STI bound to β-tryptase in a crystal structure.\n3) There is an epitope binning BLI experiment confirming the E82.AS epitope does not overlap\nwith 31A.v1110 and E104.v2 mentioned as “data not shown” – would the authors consider\nadding this data the the supplement?\nAuthors: We thank the reviewer for this suggestion. While it is clear that the epitopes are very\ndifferent based on the cryoEM structure of the complex, we agree with the reviewer that it is\nbetter to show the data for the BLI binning experiment. We have carried out additional\nexperiments and include that data as a new Supplementary Fig. S1 and replaced “(data not\nshown)” with “(Supplementary Fig. S1)”. We have renumbered Supplementary Figs S1-S7 as\nSupplementary Figs S2-S8 in the text and the Supplementary Figures.\n4) Would the authors please check if this sentence in the discussion, “Increasing potency of\nE82.AS, by introducing the double mutant HC.S30R.Y31R, did not rescue the loss of inhibitory\nactivity created by the LC.D93G mutation, indicating that LC.D93 is the key driver of the\nallosteric inhibition (Fig. 4b)” is referring to Sup Fig 8? In any case, I suggest moving S8 to the\nmain figures.\nAuthors: We thank the reviewer very much for pointing out this important observation. Indeed\nFig. 4b in this particular sentence does refer to Supplementary Fig. S8. We have modified the\npaper to replace 4b with 4c in the main text where appropriate and we have moved\nthe Fig. 4 legend with the legend from Supplementary Fig. S8.\n5) The authors should add the atomic model for the nanobody to the pdb. It has reasonable\ndensity, and based it being referenced, I believe its sequence is published. The stated rationale\nfor its omission is that it “was used solely as a fiducial marker for data processing” which would\nalso apply to the Fab E104.v2 that was included in the model.\nAuthors: We thank the reviewer for this suggestion. We have added the nanobody coordinates\nto the pdb as requested. We have deleted the original text:\nOriginal text: “The nanobody was used solely as a fiducial marker for data processing and was\nnot modeled.”\nNew text: “The nanobody was rigid fit into the density from the crystal structure PDB: 6ANA.\nBecause of the low resolution of the map in this region, side chains were not assigned.”\nOriginal text (Fig. 2 legend): “The structure of the V H nanobody (Nb), which served as a\nH\nfiducial marker, was not modeled.”\nNew text (Fig. 2 legend): The structure of the V H nanobody (Nb) in grey, which served as a\nH\nfiducial marker, was rigid fit into the density from the crystal structure PDB: 6ANA.\nUpon reviewing this comment, we realized that we never described the nanobody in the\nexperimental. We apologize for this and now provide new text to the Methods section.\nNew text: “Generation and purification of anti--tryptase antibodies and the anti-kappa\nnanobody”\nNew text: “The nanobody was produced in 293 cells by transient transfection and purified using\nstandard Ni-affinity chromatography followed by size exclusion chromatography using an S-75\ncolumn in 20 mM histidine acetate pH 5.5, 150 mM NaCl buffer.”\n6) If possible, I recommend the authors deposit the raw cryo-EM movies to EMPIAR.\nAuthors: We have deposited the raw cryo-EM movies to EMPIAR as recommended by the\nreviewer. We have included this statement “Raw cryo-EM movies have been deposited in\nEMPIAR74 as EMPIAR-13094.” in the methods section and the data availability section. We have\nadded a citation to EMPIAR (New ref 74) per that journal request. 74. Iudin, A. et al. EMPIAR:\nthe electron microscopy public image archive. Nucleic Acids Res, 51, D1503-D1511 (2023).\nReviewer #4 (Remarks to the Author):\npart of the Nature Communications initiative to facilitate training in peer review and to provide\nappropriate recognition for Early Career Researchers who co-review manuscripts.\nAuthors: We thank this Early Career Researcher for their comments that were included in one\nof the reviews above.\nWe also modified the format for the references to conform to Nature Communications style.\nResponse to Referees\nReviewer #2 (Remarks to the Author)\nI thank the authors for addressing my main comments.\nThe authors state that they have \"annotated important regions in the revised Supplementary File\nSI_HXPFexcel.xlsx. Namely, we have highlighted residues that are within 4 Å of antibody E82.AS as seen\nin the cryo-EM structure...\".\nI cannot see this annotation in the version of the .xlsx I can download of the named file. Please recheck.\nAlso, I still think it would benefit the ability of a reader to assess the data and its quality if they authors\nprovided some examples of HDX uptake curves in the Supporting Information of important regions - like I\nspecify in my initial comments.\nReviewer #3 (Remarks to the Author):\nThe authors have addressed all my suggestions. I congratulate them on this exciting study.\n- Dan Kober\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nAuthor Response to Reviewer #2\nWe thank the reviewer for carefully inspecting our HDX data that we submitted along with the\nmanuscript. We apologize for the confusion we caused by uploading an earlier version of Supplementary\nFile SI_HXPFexcel.xlsx instead of the corrected one.\nWe now provide the correct version for Supplementary File SI_HXPFexcel.xlsx. In this version, the tab\nnamed “smallest peptide empirical PF” shows colored tryptase residues that are within 4 Å of the E82.AS\nFab (light blue), the catalytic triad residues (orange), residues that contain the disulfide bond connecting\nthe 180s and 220s loop (yellow) and other key residues in the active site (green), which are not in\ncontact with the Fab so that the residues and their corresponding HX data can easily be correlated in the\nexcel file.\nRegarding the request to provide some examples of HDX uptake curves, we point out that the HDX\nuptake curves of not only the important regions, but all the peptides, are shown in Supplementary Figure\n12.\nAuthor Response to Reviewer #3\nWe thank this reviewer for their congratulations and are pleased to know they feel this is an exciting\nstudy.\nAuthor Response to Reviewer #4\nWe thank this reviewer for their time and input."}, {"round": 2, "reviewer_comments": {"reviewer_2": "I thank the authors for addressing my main comments.\nThe authors state that they have \"annotated important regions in the revised\nSupplementary File SI_HXPFexcel.xlsx. Namely, we have highlighted residues that are within 4 Å\nof antibody E82.AS as seen in the cryo-EM structure... \".\nI cannot see this annotation in the version of the .xlsx I can download of the named file. Please recheck.\nAlso, I still think it would benefit the ability of a reader to assess the data and its quality if they authors provided some\nexamples of HDX uptake curves in the Supporting Information of important regions - like I specify in my initial comments.", "reviewer_3": "The authors have addressed all my suggestions. I congratulate them on this exciting study.\n- Dan Kober", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Author Response to Reviewer #2\nWe thank the reviewer for carefully inspecting our HDX data that we submitted along with the\nmanuscript. We apologize for the confusion we caused by uploading an earlier version of Supplementary\nFile SI_HXPFexcel.xlsx instead of the corrected one.\nWe now provide the correct version for Supplementary File SI_HXPFexcel.xlsx. In this version, the tab\nnamed “smallest peptide empirical PF” shows colored tryptase residues that are within 4 Å of the E82.AS\nFab (light blue), the catalytic triad residues (orange), residues that contain the disulfide bond connecting\nthe 180s and 220s loop (yellow) and other key residues in the active site (green), which are not in\ncontact with the Fab so that the residues and their corresponding HX data can easily be correlated in the\nexcel file.\nRegarding the request to provide some examples of HDX uptake curves, we point out that the HDX\nuptake curves of not only the important regions, but all the peptides, are shown in Supplementary Figure\n12.\nAuthor Response to Reviewer #3\nWe thank this reviewer for their congratulations and are pleased to know they feel this is an exciting\nstudy.\nAuthor Response to Reviewer #4\nWe thank this reviewer for their time and input."}]

Decision letter