Consequences of kelp forest ecosystem shifts and predictors of persistence through multiple stressors.

Ecological communities can be stable over multiple generations, or rapidly shift into structurally and functionally different configurations. In kelp forest ecosystems, overgrazing by sea urchins can abruptly shift forests into alternative states that are void of macroalgae and primarily dominated by actively grazing sea urchins. Beginning in 2014, a sea urchin outbreak along the central coast of California resulted in a patchy mosaic of remnant forests interspersed with sea urchin barrens. In this study, we used a 14-year subtidal monitoring dataset of invertebrates, algae, and fishes to explore changes in community structure associated with the loss of forests. We found that the spatial mosaic of barrens and forests resulted in a region-wide shift in community structure. However, the magnitude of kelp forest loss and taxonomic-level consequences were spatially heterogeneous. Taxonomic diversity declined across the region, but there were no declines in richness for any group, suggesting compositional redistribution. Baseline ecological and environmental conditions, and sea urchin behaviour, explained the persistence of forests through multiple stressors. These results indicate that spatial heterogeneity in preexisting ecological and environmental conditions can explain patterns of community change.

Identification and characterisation of anti-IL-13 inhibitory single domain antibodies provides new insights into receptor selectivity and attractive opportunities for drug discovery.

Interleukin-13 (IL-13) is a cytokine involved in T-cell immune responses and is a well validated therapeutic target for the treatment of asthma, along with other allergic and inflammatory diseases. IL-13 signals through a ternary signalling complex formed with the receptors IL-13Rα1 and IL-4Rα. This complex is assembled by IL-13 initially binding IL-13Rα1, followed by association of the binary IL-13:IL-13Rα1 complex with IL-4Rα. The receptors are shared with IL-4, but IL-4 initially binds IL-4Rα. Here we report the identification and characterisation of a diverse panel of single-domain antibodies (VHHs) that bind to IL-13 (KD 40 nM-5.5 µM) and inhibit downstream IL-13 signalling (IC50 0.2-53.8 µM). NMR mapping showed that the VHHs recognise a number of epitopes on IL-13, including previously unknown allosteric sites. Further NMR investigation of VHH204 bound to IL-13 revealed a novel allosteric mechanism of inhibition, with the antibody stabilising IL-13 in a conformation incompatible with receptor binding. This also led to the identification of a conformational equilibrium for free IL-13, providing insights into differing receptor signalling complex assembly seen for IL-13 compared to IL-4, with formation of the IL-13:IL-13Rα1 complex required to stabilise IL-13 in a conformation with high affinity for IL-4Rα. These findings highlight new opportunities for therapeutic targeting of IL-13 and we report a successful 19F fragment screen of the IL-13:VHH204 complex, including binding sites identified for several hits. To our knowledge, these 19F containing fragments represent the first small-molecules shown to bind to IL-13 and could provide starting points for a small-molecule drug discovery programme.

A marine protected area network does not confer community structure resilience to a marine heatwave across coastal ecosystems.

Marine protected areas (MPAs) have gained attention as a conservation tool for enhancing ecosystem resilience to climate change. However, empirical evidence explicitly linking MPAs to enhanced ecological resilience is limited and mixed. To better understand whether MPAs can buffer climate impacts, we tested the resistance and recovery of marine communities to the 2014-2016 Northeast Pacific heatwave in the largest scientifically designed MPA network in the world off the coast of California, United States. The network consists of 124 MPAs (48 no-take state marine reserves, and 76 partial-take or special regulation conservation areas) implemented at different times, with full implementation completed in 2012. We compared fish, benthic invertebrate, and macroalgal community structure inside and outside of 13 no-take MPAs across rocky intertidal, kelp forest, shallow reef, and deep reef nearshore habitats in California's Central Coast region from 2007 to 2020. We also explored whether MPA features, including age, size, depth, proportion rock, historic fishing pressure, habitat diversity and richness, connectivity, and fish biomass response ratios (proxy for ecological performance), conferred climate resilience for kelp forest and rocky intertidal habitats spanning 28 MPAs across the full network. Ecological communities dramatically shifted due to the marine heatwave across all four nearshore habitats, and MPAs did not facilitate habitat-wide resistance or recovery. Only in protected rocky intertidal habitats did community structure significantly resist marine heatwave impacts. Community shifts were associated with a pronounced decline in the relative proportion of cold water species and an increase in warm water species. MPA features did not explain resistance or recovery to the marine heatwave. Collectively, our findings suggest that MPAs have limited ability to mitigate the impacts of marine heatwaves on community structure. Given that mechanisms of resilience to climate perturbations are complex, there is a clear need to expand assessments of ecosystem-wide consequences resulting from acute climate-driven perturbations, and the potential role of regulatory protection in mitigating community structure changes.

Modulation of IL-17 backbone dynamics reduces receptor affinity and reveals a new inhibitory mechanism.

Knowledge of protein dynamics is fundamental to the understanding of biological processes, with NMR and 2D-IR spectroscopy being two of the principal methods for studying protein dynamics. Here, we combine these two methods to gain a new understanding of the complex mechanism of a cytokine:receptor interaction. The dynamic nature of many cytokines is now being recognised as a key property in the signalling mechanism. Interleukin-17s (IL-17) are proinflammatory cytokines which, if unregulated, are associated with serious autoimmune diseases such as psoriasis, and although there are several therapeutics on the market for these conditions, small molecule therapeutics remain elusive. Previous studies, exploiting crystallographic methods alone, have been unable to explain the dramatic differences in affinity observed between IL-17 dimers and their receptors, suggesting there are factors that cannot be fully explained by the analysis of static structures alone. Here, we show that the IL-17 family of cytokines have varying degrees of flexibility which directly correlates to their receptor affinities. Small molecule inhibitors of the cytokine:receptor interaction are usually thought to function by either causing steric clashes or structural changes. However, our results, supported by other biophysical methods, provide evidence for an alternate mechanism of inhibition, in which the small molecule rigidifies the protein, causing a reduction in receptor affinity. The results presented here indicate an induced fit model of cytokine:receptor binding, with the more flexible cytokines having a higher affinity. Our approach could be applied to other systems where the inhibition of a protein-protein interaction has proved intractable, for example due to the flat, featureless nature of the interface. Targeting allosteric sites which modulate protein dynamics, opens up new avenues for novel therapeutic development.

Co-crystallisation and humanisation of an anti-HER2 single-domain antibody as a theranostic tool.

Human epidermal growth factor receptor-2 (HER2) is a well-recognised biomarker associated with 25% of breast cancers. In most cases, early detection and/or treatment correlates with an increased chance of survival. This study, has identified and characterised a highly specific anti-HER2 single-domain antibody (sdAb), NM-02, as a potential theranostic tool. Complete structural description by X-ray crystallography has revealed a non-overlapping epitope with current anti-HER2 antibodies. To reduce the immunogenicity risk, NM-02 underwent a humanisation process and retained wild type-like binding properties. To further de-risk the progression towards chemistry, manufacturing and control (CMC) we performed full developability profiling revealing favourable thermal and physical biochemical 'drug-like' properties. Finally, the application of the lead humanised NM-02 candidate (variant K) for HER2-specific imaging purposes was demonstrated using breast cancer HER2+/BT474 xenograft mice.

Nanobody inhibitors of Plexin-B1 identify allostery in plexin-semaphorin interactions and signaling.

Plexin-B1 is a receptor for the cell surface semaphorin, Sema4D. This signaling system has been implicated in a variety of human diseases, including cancer, multiple sclerosis and osteoporosis. While inhibitors of the Plexin-B1:Sema4D interaction have been previously reported, understanding their mechanism has been hindered by an incomplete structural view of Plexin-B1. In this study, we have raised and characterized a pair of nanobodies that are specific for mouse Plexin-B1 and which inhibit the binding of Sema4D to mouse Plexin-B1 and its biological activity. Structural studies of these nanobodies reveal that they inhibit the binding of Sema4D in an allosteric manner, binding to epitopes not previously reported. In addition, we report the first unbound structure of human Plexin-B1, which reveals that Plexin-B1 undergoes a conformational change on Sema4D binding. These changes mirror those seen upon binding of allosteric peptide modulators, which suggests a new model for understanding Plexin-B1 signaling and provides a potential innovative route for therapeutic modulation of Plexin-B1.

Identification, binding, and structural characterization of single domain anti-PD-L1 antibodies inhibitory of immune regulatory proteins PD-1 and CD80.

Programmed death-ligand 1 (PD-L1) is a key immune regulatory protein that interacts with programmed cell death protein 1 (PD-1), leading to T-cell suppression. Whilst this interaction is key in self-tolerance, cancer cells evade the immune system by overexpressing PD-L1. Inhibition of the PD-1/PD-L1 pathway with standard monoclonal antibodies has proven a highly effective cancer treatment; however, single domain antibodies (VHH) may offer numerous potential benefits. Here, we report the identification and characterization of a diverse panel of 16 novel VHHs specific to PD-L1. The panel of VHHs demonstrate affinities of 0.7 nM to 5.1 µM and were able to completely inhibit PD-1 binding to PD-L1. The binding site for each VHH on PD-L1 was determined using NMR chemical shift perturbation mapping and revealed a common binding surface encompassing the PD-1-binding site. Additionally, we solved crystal structures of two representative VHHs in complex with PD-L1, which revealed unique binding modes. Similar NMR experiments were used to identify the binding site of CD80 on PD-L1, which is another immune response regulatory element and interacts with PD-L1 localized on the same cell surface. CD80 and PD-1 were revealed to share a highly overlapping binding site on PD-L1, with the panel of VHHs identified expected to inhibit CD80 binding. Comparison of the CD80 and PD-1 binding sites on PD-L1 enabled the identification of a potential antibody binding region able to confer specificity for the inhibition of PD-1 binding only, which may offer therapeutic benefits to counteract cancer cell evasion of the immune system.

Multistressor global change drivers reduce hatch and viability of Lingcod embryos, a benthic egg layer in the California Current System.

Early life history stages of marine fishes are often more susceptible to environmental stressors than adult stages. This vulnerability is likely exacerbated for species that lay benthic egg masses bound to substrate because the embryos cannot evade locally unfavorable environmental conditions. Lingcod (Ophiodon elongatus), a benthic egg layer, is an ecologically and economically significant predator in the highly-productive California Current System (CCS). We ran a flow-through mesocosm experiment that exposed Lingcod eggs collected from Monterey Bay, CA to conditions we expect to see in the central CCS by the year 2050 and 2100. Exposure to temperature, pH, and dissolved oxygen concentrations projected by the year 2050 halved the successful hatch of Lingcod embryos and significantly reduced the size of day-1 larvae. In the year 2100 treatment, viable hatch plummeted (3% of normal), larvae were undersized (83% of normal), yolk reserves were exhausted (38% of normal), and deformities were widespread (94% of individuals). This experiment is the first to expose marine benthic eggs to future temperature, pH, and dissolved oxygen conditions in concert. Lingcod are a potential indicator species for other benthic egg layers for which global change conditions may significantly diminish recruitment rates.

Sequence-specific 1H, 13C and 15N backbone NMR assignments for the N-terminal IgV-like domain (D1) and full extracellular region (D1D2) of PD-L1.

The co-inhibitory immune checkpoint interaction between programmed cell death-protein 1 (PD-1) and programmed cell death-ligand 1 (PD-L1) serves to regulate T-cell activation, promoting self-tolerance. Over-expression of PD-L1 is a mechanism through which tumour cells can evade detection by the immune system. Several therapeutic antibodies targeting PD-L1 or PD-1 have been approved for the treatment of a variety of cancers, however, the discovery and development of small-molecule inhibitors of PD-L1 remains a challenge. Here we report comprehensive sequence-specific backbone resonance assignments (1H, 13C, and 15N) obtained for the N-terminal IgV-like domain of PD-L1 (D1) and the full two domain extracellular region (D1D2). These NMR assignments will serve as a useful tool in the discovery of small-molecule therapeutics targeting PD-L1 and in the characterisation of functional interactions with other protein partners, such as CD80.

Large-scale, multidecade monitoring data from kelp forest ecosystems in California and Oregon (USA).

Kelp forests are among the most productive ecosystems on Earth. In combination with their close proximity to the shore, the productivity and biodiversity of these ecosystems generate a wide range of ecosystem services including supporting (e.g., primary production, habitat), regulating (e.g., water flow, coastal erosion), provisioning (e.g., commercial and recreational fisheries), and cultural (e.g., recreational, artisanal) services. For these reasons, kelp forests have long been the target of ecological studies. However, with few exceptions, these studies have been localized and short term (<5 years). In 1999, recognizing the importance of large-scale, long-term studies for understanding the structure, functioning, and dynamics of coastal marine ecosystems, and for informing policy, the Partnership for Interdisciplinary Studies of Coastal Oceans (PISCO) designed and initiated a large-scale, long-term monitoring study of kelp forest ecosystems along 1400 km of coast stretching from southern California to southern Oregon, USA. The purpose of the study has been to characterize the spatial and temporal patterns of kelp forest ecosystem structure and evaluate the relative contributions of biological and environmental variables derived from external sources (e.g., sea otter density, Chl-a concentration, sea surface temperature, wave energy) in explaining observed spatial and temporal patterns. For this purpose, the ecological community (i.e., density, percent cover, or biomass of conspicuous fishes, invertebrates, and macroalgae) and geomorphological attributes (bottom depth, substratum type, and vertical relief) of kelp forest ecosystems have been surveyed annually using SCUBA divers trained in both scientific diving and data collection techniques and the identification of kelp forest species. The study region spans distinct ecological and biogeographic provinces, which enables investigations of how variation in environmental drivers and distinctive species compositions influence community structure, and its response to climate-related environmental change across a portion of the California Current Large Marine Ecosystem. These data have been used to inform fisheries management, design and evaluate California's state-wide network of marine protected areas (MPAs), and assess the ecological consequences of climate change (e.g., marine heatwaves). Over time, the spatial and temporal design of the monitoring program was adapted to fill its role in evaluating the ecological responses to the establishment of MPAs. There are no copyright restrictions; please cite this paper when data are used.

Discovery of a novel pseudo ß-hairpin structure of N-truncated amyloid-ß for use as a vaccine against Alzheimer's disease.

One of the hallmarks of Alzheimer's disease (AD) are deposits of amyloid-beta (Aß) protein in amyloid plaques in the brain. The Aß peptide exists in several forms, including full-length Aß1-42 and Aß1-40 - and the N-truncated species, pyroglutamate Aß3-42 and Aß4-42, which appear to play a major role in neurodegeneration. We previously identified a murine antibody (TAP01), which binds specifically to soluble, non-plaque N-truncated Aß species. By solving crystal structures for TAP01 family antibodies bound to pyroglutamate Aß3-14, we identified a novel pseudo ß-hairpin structure in the N-terminal region of Aß and show that this underpins its unique binding properties. We engineered a stabilised cyclic form of Aß1-14 (N-Truncated Amyloid Peptide AntibodieS; the 'TAPAS' vaccine) and showed that this adopts the same 3-dimensional conformation as the native sequence when bound to TAP01. Active immunisation of two mouse models of AD with the TAPAS vaccine led to a striking reduction in amyloid-plaque formation, a rescue of brain glucose metabolism, a stabilisation in neuron loss, and a rescue of memory deficiencies. Treating both models with the humanised version of the TAP01 antibody had similar positive effects. Here we report the discovery of a unique conformational epitope in the N-terminal region of Aß, which offers new routes for active and passive immunisation against AD.

Cross-Reactive SARS-CoV-2 Neutralizing Antibodies From Deep Mining of Early Patient Responses.

Passive immunization using monoclonal antibodies will play a vital role in the fight against COVID-19. The recent emergence of viral variants with reduced sensitivity to some current antibodies and vaccines highlights the importance of broad cross-reactivity. This study describes deep-mining of the antibody repertoires of hospitalized COVID-19 patients using phage display technology and B cell receptor (BCR) repertoire sequencing to isolate neutralizing antibodies and gain insights into the early antibody response. This comprehensive discovery approach has yielded a panel of potent neutralizing antibodies which bind distinct viral epitopes including epitopes conserved in SARS-CoV-1. Structural determination of a non-ACE2 receptor blocking antibody reveals a previously undescribed binding epitope, which is unlikely to be affected by the mutations in any of the recently reported major viral variants including B.1.1.7 (from the UK), B.1.351 (from South Africa) and B.1.1.28 (from Brazil). Finally, by combining sequences of the RBD binding and neutralizing antibodies with the B cell receptor repertoire sequencing, we also describe a highly convergent early antibody response. Similar IgM-derived sequences occur within this study group and also within patient responses described by multiple independent studies published previously.

Conformational dynamics in interleukin 17A and 17F functional complexes is a key determinant of receptor A affinity and specificity.

The proinflammatory cytokines IL-17A and IL-17F have been identified as key drivers of a range of human inflammatory diseases, such as psoriasis, which has led to several therapeutic antibodies targeted at IL-17A. The two cytokines have been shown to tightly associate as functional homo and hetero dimers, which induce signalling via the formation of a cell surface signalling complex with a single copy of both IL-17RA and IL-17RC. Striking differences in affinity have been observed for IL-17RA binding to IL-17AA, IL-17AF and IL-17FF, however, the functional significance and molecular basis for this has remained unclear. We have obtained comprehensive backbone NMR assignments for full length IL-17AA (79%), IL-17AF (93%) and IL-17FF (89%), which show that the dimers adopt almost identical backbone topologies in solution to those observed in reported crystal structures. Analysis of the line widths and intensities of assigned backbone amide NMR signals has revealed striking differences in the conformational plasticity and dynamics of IL-17AA compared to both IL-17AF and IL-17FF. Our NMR data indicate that a number of regions of IL-17AA are interconverting between at least two distinct conformations on a relatively slow timescale. Such conformational heterogeneity has previously been shown to play an important role in the formation of many high affinity protein-protein complexes. The locations of the affected IL-17AA residues essentially coincides with the regions of both IL-17A and IL-17F previously shown to undergo significant structural changes on binding to IL-17RA. Substantially less conformational exchange was revealed by the NMR data for IL-17FF and IL-17AF. We propose that the markedly different conformational dynamic properties of the distinct functional IL-17 dimers plays a key role in determining their affinities for IL-17RA, with the more dynamic and plastic nature of IL-17AA contributing to the significantly tighter affinity observed for binding to IL-17RA. In contrast, the dynamic properties are expected to have little influence on the affinity of IL-17 dimers for IL-17RC, which has recently been shown to induce only small structural changes in IL-17FF upon binding.

Large-scale shift in the structure of a kelp forest ecosystem co-occurs with an epizootic and marine heatwave.

Climate change is responsible for increased frequency, intensity, and duration of extreme events, such as marine heatwaves (MHWs). Within eastern boundary current systems, MHWs have profound impacts on temperature-nutrient dynamics that drive primary productivity. Bull kelp (Nereocystis luetkeana) forests, a vital nearshore habitat, experienced unprecedented losses along 350 km of coastline in northern California beginning in 2014 and continuing through 2019. These losses have had devastating consequences to northern California communities, economies, and fisheries. Using a suite of in situ and satellite-derived data, we demonstrate that the abrupt ecosystem shift initiated by a multi-year MHW was preceded by declines in keystone predator population densities. We show strong evidence that northern California kelp forests, while temporally dynamic, were historically resilient to fluctuating environmental conditions, even in the absence of key top predators, but that a series of coupled environmental and biological shifts between 2014 and 2016 resulted in the formation of a persistent, altered ecosystem state with low primary productivity. Based on our findings, we recommend the implementation of ecosystem-based and adaptive management strategies, such as (1) monitoring the status of key ecosystem attributes: kelp distribution and abundance, and densities of sea urchins and their predators, (2) developing management responses to threshold levels of these attributes, and (3) creating quantitative restoration suitability indices for informing kelp restoration efforts.

Geographic variation in responses of kelp forest communities of the California Current to recent climatic changes.

The changing global climate is having profound effects on coastal marine ecosystems around the world. Structure, functioning, and resilience, however, can vary geographically, depending on species composition, local oceanographic forcing, and other pressures from human activities and use. Understanding ecological responses to environmental change and predicting changes in the structure and functioning of whole ecosystems require large-scale, long-term studies, yet most studies trade spatial extent for temporal duration. We address this shortfall by integrating multiple long-term kelp forest monitoring datasets to evaluate biogeographic patterns and rates of change of key functional groups (FG) along the west coast of North America. Analysis of data from 469 sites spanning Alaska, USA, to Baja California, Mexico, and 373 species (assigned to 18 FG) reveals regional variation in responses to both long-term (2006-2016) change and a recent marine heatwave (2014-2016) associated with two atmospheric and oceanographic anomalies, the "Blob" and extreme El Niño Southern Oscillation (ENSO). Canopy-forming kelps appeared most sensitive to warming throughout their range. Other FGs varied in their responses among trophic levels, ecoregions, and in their sensitivity to heatwaves. Changes in community structure were most evident within the southern and northern California ecoregions, while communities in the center of the range were more resilient. We report a poleward shift in abundance of some key FGs. These results reveal major, ongoing region-wide changes in productive coastal marine ecosystems in response to large-scale climate variability, and the potential loss of foundation species. In particular, our results suggest that coastal communities that are dependent on kelp forests will be more impacted in the southern portion of the California Current region, highlighting the urgency of implementing adaptive strategies to sustain livelihoods and ensure food security. The results also highlight the value of multiregional integration and coordination of monitoring programs for improving our understanding of marine ecosystems, with the goal of informing policy and resource management in the future.

Structural and functional characterization of C0021158, a high-affinity monoclonal antibody that inhibits Arginase 2 function via a novel non-competitive mechanism of action.

Arginase 2 (ARG2) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine. The dysregulated expression of ARG2 within specific tumor microenvironments generates an immunosuppressive niche that effectively renders the tumor 'invisible' to the host's immune system. Increased ARG2 expression leads to a concomitant depletion of local L-arginine levels, which in turn leads to suppression of anti-tumor T-cell-mediated immune responses. Here we describe the isolation and characterization of a high affinity antibody (C0021158) that inhibits ARG2 enzymatic function completely, effectively restoring T-cell proliferation in vitro. Enzyme kinetic studies confirmed that C0021158 exhibits a noncompetitive mechanism of action, inhibiting ARG2 independently of L-arginine concentrations. To elucidate C0021158's inhibitory mechanism at a structural level, the co-crystal structure of the Fab in complex with trimeric ARG2 was solved. C0021158's epitope was consequently mapped to an area some distance from the enzyme's substrate binding cleft, indicating an allosteric mechanism was being employed. Following C0021158 binding, distinct regions of ARG2 undergo major conformational changes. Notably, the backbone structure of a surface-exposed loop is completely rearranged, leading to the formation of a new short helix structure at the Fab-ARG2 interface. Moreover, this large-scale structural remodeling at ARG2's epitope translates into more subtle changes within the enzyme's active site. An arginine residue at position 39 is reoriented inwards, sterically impeding the binding of L-arginine. Arg39 is also predicted to alter the pKA of a key catalytic histidine residue at position 160, further attenuating ARG2's enzymatic function. In silico molecular docking simulations predict that L-arginine is unable to bind effectively when antibody is bound, a prediction supported by isothermal calorimetry experiments using an L-arginine mimetic. Specifically, targeting ARG2 in the tumor microenvironment through the application of C0021158, potentially in combination with standard chemotherapy regimens or alternate immunotherapies, represents a potential new strategy to target immune cold tumors.

Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.

Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity.

Allosteric activation of MALT1 by its ubiquitin-binding Ig3 domain.

The catalytic activity of the protease MALT1 is required for adaptive immune responses and regulatory T (Treg)-cell development, while dysregulated MALT1 activity can lead to lymphoma. MALT1 activation requires its monoubiquitination on lysine 644 (K644) within the Ig3 domain, localized adjacent to the protease domain. The molecular requirements for MALT1 monoubiquitination and the mechanism by which monoubiquitination activates MALT1 had remained elusive. Here, we show that the Ig3 domain interacts directly with ubiquitin and that an intact Ig3-ubiquitin interaction surface is required for the conjugation of ubiquitin to K644. Moreover, by generating constitutively active MALT1 mutants that overcome the need for monoubiquitination, we reveal an allosteric communication between the ubiquitination site K644, the Ig3-protease interaction surface, and the active site of the protease domain. Finally, we show that MALT1 mutants that alter the Ig3-ubiquitin interface impact the biological response of T cells. Thus, ubiquitin binding by the Ig3 domain promotes MALT1 activation by an allosteric mechanism that is essential for its biological function.