Paths reunited: initiation of the classical and lectin pathways of complement activation

Understanding the structural organisation and mode of action of the initiating complex of the classical pathway of complement activation (C1) has been a central goal in complement biology since its isolation almost 50 years ago. Nevertheless, knowledge is still incomplete, especially with regard to the interactions between its subcomponents C1q, C1r and C1s that trigger activation upon binding to a microbial target. Recent studies have provided new insights into these interactions, and have revealed unexpected parallels with initiating complexes of the lectin pathway of complement: MBL–MASP and ficolin–MASP. Here, we develop and expand these concepts and delineate their implications towards the key aspects of complement activation via the classical and lectin pathways

The cytotoxic domain of colicin E9 is a channel-forming endonuclease

Bacterial toxins commonly translocate cytotoxic enzymes into cells using dedicated channelforming subunits or domains as conduits. We demonstrate that the small cytotoxic endonuclease domain from the bacterial toxin colicin E9 (the E9 DNase) exhibits nonvoltage- gated, channel-forming activity in planar lipid bilayers and that this activity is linked to toxin translocation into cells. A disulfide bond engineered into the DNase abolished channel activity and colicin toxicity but left endonuclease activity unaffected, with NMR experiments suggesting decreased conformational flexibility as the likely reason for these alterations. Concomitant with the reduction of the disulfide bond was the restoration of conformational flexibility, DNase channel activity and colicin toxicity. Our data suggest that endonuclease domains of colicins may mediate their own translocation across the bacterial inner membrane through an intrinsic channel activity that is dependent on structural plasticity in the protein

Engineering novel complement activity into a pulmonary surfactant protein

Complement neutralizes invading pathogens, stimulates inflammatory and adaptive immune responses, and targets non- or altered-self structures for clearance. In the classical and lectin activation pathways, it is initiated when complexes composed of separate recognition and activation subcomponents bind to a pathogen surface. Despite its apparent complexity, recognition-mediated activation has evolved independently in three separate protein families, C1q, mannose-binding lectins (MBLs), and serum ficolins. Although unrelated, all have bouquet-like architectures and associate with complement-specific serine proteases: MBLs and ficolins with MBL-associated serine protease-2 (MASP-2) and C1q with C1r and C1s. To examine the structural requirements for complement activation, we have created a number of novel recombinant rat MBLs in which the position and orientation of the MASP-binding sites have been changed. We have also engineered MASP binding into a pulmonary surfactant protein (SP-A), which has the same domain structure and architecture as MBL but lacks any intrinsic complement activity. The data reveal that complement activity is remarkably tolerant to changes in the size and orientation of the collagenous stalks of MBL, implying considerable rotational and conformational flexibility in unbound MBL. Furthermore, novel complement activity is introduced concurrently with MASP binding in SP-A but is uncontrolled and occurs even in the absence of a carbohydrate target. Thus, the active rather than the zymogen state is default in lectin·MASP complexes and must be inhibited through additional regions in circulating MBLs until triggered by pathogen recognition

Proactive vaccination using multiviral Quartet Nanocages to elicit broad anti-coronavirus responses

Defending against future pandemics requires vaccine platforms that protect across a range of related pathogens. Nanoscale patterning can be used to address this issue. Here, we produce quartets of linked receptor-binding domains (RBDs) from a panel of SARS-like betacoronaviruses, coupled to a computationally designed nanocage through SpyTag/SpyCatcher links. These Quartet Nanocages, possessing a branched morphology, induce a high level of neutralizing antibodies against several different coronaviruses, including against viruses not represented in the vaccine. Equivalent antibody responses are raised to RBDs close to the nanocage or at the tips of the nanoparticle’s branches. In animals primed with SARS-CoV-2 Spike, boost immunizations with Quartet Nanocages increase the strength and breadth of an otherwise narrow immune response. A Quartet Nanocage including the Omicron XBB.1.5 ‘Kraken’ RBD induced antibodies with binding to a broad range of sarbecoviruses, as well as neutralizing activity against this variant of concern. Quartet nanocages are a nanomedicine approach with potential to confer heterotypic protection against emergent zoonotic pathogens and facilitate proactive pandemic protection

Design and Evolution of Enhanced Peptide-Peptide Ligation for Modular Transglutaminase Assembly.

Robust and precise tools are needed to enhance the functionality and resilience of synthetic nanoarchitectures. Here, we have employed directed evolution and rational design to build a fast-acting molecular superglue from a bacterial adhesion protein. We have generated the SnoopLigase2 coupling system, a genetically encoded route for efficient transamidation between SnoopTag2 and DogTag2 peptides. Each peptide was selected for rapid reaction by phage display screening. The optimized set allows more than 99% completion and is compatible with diverse buffers, pH values, and temperatures, accelerating the reaction over 1000-fold. SnoopLigase2 directs a specific reaction in the mammalian secretory pathway, allowing covalent display on the plasma membrane. Transglutaminase 2 (TG2) has a network of interactions and substrates amidst the mammalian cell surface and extracellular matrix. We expressed a modified TG2 with resistance to oxidative inactivation and minimal self-reactivity. SnoopLigase2 enables TG2 functionalization with transforming growth factor alpha (TGFα) in routes that would be impossible through genetic fusion. The TG2:TGFα conjugate retained transamidase activity, stably anchored TGFα for signal activation in the extracellular environment, and reprogrammed cell behavior. This modular toolbox should create new opportunities for molecular assembly, both for novel biomaterials and complex cellular environments

Design and Evolution of Enhanced Peptide–Peptide Ligation for Modular Transglutaminase Assembly

Robust and precise tools are needed to enhance the functionality and resilience of synthetic nanoarchitectures. Here, we have employed directed evolution and rational design to build a fast-acting molecular superglue from a bacterial adhesion protein. We have generated the SnoopLigase2 coupling system, a genetically encoded route for efficient transamidation between SnoopTag2 and DogTag2 peptides. Each peptide was selected for rapid reaction by phage display screening. The optimized set allows more than 99% completion and is compatible with diverse buffers, pH values, and temperatures, accelerating the reaction over 1000-fold. SnoopLigase2 directs a specific reaction in the mammalian secretory pathway, allowing covalent display on the plasma membrane. Transglutaminase 2 (TG2) has a network of interactions and substrates amidst the mammalian cell surface and extracellular matrix. We expressed a modified TG2 with resistance to oxidative inactivation and minimal self-reactivity. SnoopLigase2 enables TG2 functionalization with transforming growth factor alpha (TGFα) in routes that would be impossible through genetic fusion. The TG2:TGFα conjugate retained transamidase activity, stably anchored TGFα for signal activation in the extracellular environment, and reprogrammed cell behavior. This modular toolbox should create new opportunities for molecular assembly, both for novel biomaterials and complex cellular environments

SpyMask enables combinatorial assembly of bispecific binders

Abstract Bispecific antibodies are a successful and expanding therapeutic class. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or specialized formats. Here we present SpyMask, a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second SpyCatcher is protease-activatable. We engineer a panel of structurally-distinct DoubleCatchers, from which binders project in different directions. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plates. A panel of binders recognizing different HER2 epitopes were coupled to DoubleCatcher, revealing unexpected combinations with anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Bispecific activity depended sensitively on both binder orientation and DoubleCatcher scaffold geometry. These findings support the need for straightforward assembly in different formats. SpyMask provides a scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry

SpyMask enables combinatorial assembly of bispecific binders.

Bispecific antibodies are a successful and expanding therapeutic class. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or specialized formats. Here we present SpyMask, a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second SpyCatcher is protease-activatable. We engineer a panel of structurally-distinct DoubleCatchers, from which binders project in different directions. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plates. A panel of binders recognizing different HER2 epitopes were coupled to DoubleCatcher, revealing unexpected combinations with anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Bispecific activity depended sensitively on both binder orientation and DoubleCatcher scaffold geometry. These findings support the need for straightforward assembly in different formats. SpyMask provides a scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry

SpyMask enables combinatorial assembly of bispecific binders

Acknowledgements: A.H.K. and M.R.H. were funded by the Biotechnology and Biological Sciences Research Council (BBSRC BB/S007369/1). C.L.D. was supported by the BBSRC Oxford Interdisciplinary Bioscience Doctoral Training Partnership (DTP) (grant number BB/T008784/1). We thank Dr. David Staunton from the University of Oxford Department of Biochemistry Biophysical Suite for help with biophysical analysis. We thank Dr. Anthony Tumber of University of Oxford Department of Chemistry for assistance with RapidFire Mass Spectrometry, supported by the BBSRC (BB/R000344/1). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript (AAM) version arising from this submission.AbstractBispecific antibodies are a successful and expanding therapeutic class. Standard approaches to generate bispecifics are complicated by the need for disulfide reduction/oxidation or specialized formats. Here we present SpyMask, a modular approach to bispecifics using SpyTag/SpyCatcher spontaneous amidation. Two SpyTag-fused antigen-binding modules can be precisely conjugated onto DoubleCatcher, a tandem SpyCatcher where the second SpyCatcher is protease-activatable. We engineer a panel of structurally-distinct DoubleCatchers, from which binders project in different directions. We establish a generalized methodology for one-pot assembly and purification of bispecifics in 96-well plates. A panel of binders recognizing different HER2 epitopes were coupled to DoubleCatcher, revealing unexpected combinations with anti-proliferative or pro-proliferative activity on HER2-addicted cancer cells. Bispecific activity depended sensitively on both binder orientation and DoubleCatcher scaffold geometry. These findings support the need for straightforward assembly in different formats. SpyMask provides a scalable tool to discover synergy in bispecific activity, through modulating receptor organization and geometry.</jats:p