Inhibition of cleavage of human complement component C5 and the R885H C5 variant by two distinct high affinity anti-C5 nanobodies.

The human complement system plays a crucial role in immune defense. However, its erroneous activation contributes to many serious inflammatory diseases. Since most unwanted complement effector functions result from C5 cleavage into C5a and C5b, development of C5 inhibitors, such as clinically approved monoclonal antibody eculizumab, are of great interest. Here, we developed and characterized two anti-C5 nanobodies, UNbC5-1 and UNbC5-2. Using surface plasmon resonance, we determined a binding affinity of 119.9 pM for UNbC5-1 and 7.7 pM for UNbC5-2. Competition experiments determined that the two nanobodies recognize distinct epitopes on C5. Both nanobodies efficiently interfered with C5 cleavage in a human serum environment, as they prevented red blood cell lysis via membrane attack complexes (C5b-9) and the formation of chemoattractant C5a. The cryo-EM structure of UNbC5-1 and UNbC5-2 in complex with C5 (3.6 Å resolution) revealed that the binding interfaces of UNbC5-1 and UNbC5-2 overlap with known complement inhibitors eculizumab and RaCI3, respectively. UNbC5-1 binds to the MG7 domain of C5, facilitated by a hydrophobic core and polar interactions, and UNbC5-2 interacts with the C5d domain mostly by salt bridges and hydrogen bonds. Interestingly, UNbC5-1 potently binds and inhibits C5 R885H, a genetic variant of C5 that is not recognized by eculizumab. Altogether, we identified and characterized two different, high affinity nanobodies against human C5. Both nanobodies could serve as diagnostic and/or research tools to detect C5 or inhibit C5 cleavage. Furthermore, the residues targeted by UNbC5-1 hold important information for therapeutic inhibition of different polymorphic variants of C5.

Crystal structure of the second extracellular domain of human tetraspanin CD9: twinning and diffuse scattering.

Remarkable features are reported in the diffraction pattern produced by a crystal of the second extracellular domain of tetraspanin CD9 (deemed CD9EC2), the structure of which has been described previously [Oosterheert et al. (2020 ▸), Life Sci. Alliance, 3, e202000883]. CD9EC2 crystallized in space group P1 and was twinned. Two types of diffuse streaks are observed. The stronger diffuse streaks are related to the twinning and occur in the direction perpendicular to the twinning interface. It is concluded that the twin domains scatter coherently as both Bragg reflections and diffuse streaks are seen. The weaker streaks along c* are unrelated to the twinning but are caused by intermittent layers of non-crystallographic symmetry related molecules. It is envisaged that the raw diffraction images could be very useful for methods developers trying to remove the diffuse scattering to extract accurate Bragg intensities or using it to model the effect of packing disorder on the molecular structure.

Cryo-EM structures of peripherin-2 and ROM1 suggest multiple roles in photoreceptor membrane morphogenesis.

Mammalian peripherin-2 (PRPH2) and rod outer segment membrane protein 1 (ROM1) are retina-specific tetraspanins that partake in the constant renewal of stacked membrane discs of photoreceptor cells that enable vision. Here, we present single-particle cryo-electron microscopy structures of solubilized PRPH2-ROM1 heterodimers and higher-order oligomers. High-risk PRPH2 and ROM1 mutations causing blindness map to the protein-dimer interface. Cysteine bridges connect dimers forming positive-curved oligomers, whereas negative-curved oligomers were observed occasionally. Hexamers and octamers exhibit a secondary micelle that envelopes four carboxyl-terminal helices, supporting a potential role in membrane remodeling. Together, the data indicate multiple structures for PRPH2-ROM1 in creating and maintaining compartmentalization of photoreceptor cells.

Insight into mode-of-action and structural determinants of the compstatin family of clinical complement inhibitors.

With the addition of the compstatin-based complement C3 inhibitor pegcetacoplan, another class of complement targeted therapeutics have recently been approved. Moreover, compstatin derivatives with enhanced pharmacodynamic and pharmacokinetic profiles are in clinical development (e.g., Cp40/AMY-101). Despite this progress, the target binding and inhibitory modes of the compstatin family remain incompletely described. Here, we present the crystal structure of Cp40 complexed with its target C3b at 2.0-Å resolution. Structure-activity-relationship studies rationalize the picomolar affinity and long target residence achieved by lead optimization, and reveal a role for structural water in inhibitor binding. We provide explanations for the narrow species specificity of this drug class and demonstrate distinct target selection modes between clinical compstatin derivatives. Functional studies provide further insight into physiological complement activation and corroborate the mechanism of its compstatin-mediated inhibition. Our study may thereby guide the application of existing and development of next-generation compstatin analogs.

Complement component C3: A structural perspective and potential therapeutic implications.

As the most abundant component of the complement system, C3 and its proteolytic derivatives serve essential roles in the function of all three complement pathways. Central to this is a network of protein-protein interactions made possible by the sequential proteolysis and far-reaching structural changes that accompany C3 activation. Beginning with the crystal structures of C3, C3b, and C3c nearly twenty years ago, the physical transformations underlying C3 function that had long been suspected were finally revealed. In the years that followed, a compendium of crystallographic information on C3 derivatives bound to various enzymes, regulators, receptors, and inhibitors generated new levels of insight into the structure and function of the C3 molecule. This Review provides a concise classification, summary, and interpretation of the more than 50 unique crystal structure determinations for human C3. It also highlights other salient features of C3 structure that were made possible through solution-based methods, including Hydrogen/Deuterium Exchange and Small Angle X-ray Scattering. At this pivotal time when the first C3-targeted therapeutics begin to see use in the clinic, some perspectives are also offered on how this continually growing body of structural information might be leveraged for future development of next-generation C3 inhibitors.

Multifaceted Activities of Seven Nanobodies against Complement C4b.

Cleavage of the mammalian plasma protein C4 into C4b initiates opsonization, lysis, and clearance of microbes and damaged host cells by the classical and lectin pathways of the complement system. Dysregulated activation of C4 and other initial components of the classical pathway may cause or aggravate pathologies, such as systemic lupus erythematosus, Alzheimer disease, and schizophrenia. Modulating the activity of C4b by small-molecule or protein-based inhibitors may represent a promising therapeutic approach for preventing excessive inflammation and damage to host cells and tissue. Here, we present seven nanobodies, derived from llama (Lama glama) immunization, that bind to human C4b (Homo sapiens) with high affinities ranging from 3.2 nM to 14 pM. The activity of the nanobodies varies from no to complete inhibition of the classical pathway. The inhibiting nanobodies affect different steps in complement activation, in line with blocking sites for proconvertase formation, C3 substrate binding to the convertase, and regulator-mediated inactivation of C4b. For four nanobodies, we determined single-particle cryo-electron microscopy structures in complex with C4b at 3.4-4 Å resolution. The structures rationalize the observed functional effects of the nanobodies and define their mode of action during complement activation. Thus, we characterized seven anti-C4b nanobodies with diverse effects on the classical pathway of complement activation that may be explored for imaging, diagnostic, or therapeutic applications.

Improving sampling of crystallographic disorder in ensemble refinement.

Ensemble refinement, the application of molecular dynamics to crystallographic refinement, explicitly models the disorder inherent in macromolecular structures. These ensemble models have been shown to produce more accurate structures than traditional single-model structures. However, suboptimal sampling of the molecular-dynamics simulation and modelling of crystallographic disorder has limited the utility of the method, and can lead to unphysical and strained models. Here, two improvements to the ensemble refinement method implemented within Phenix are presented: DEN restraints, which guide the local sampling of conformations and allow a more robust exploration of local conformational landscapes, and ECHT disorder models, which allow the selection of more physically meaningful and effective disorder models for parameterizing the continuous disorder components within a crystal. These improvements lead to more consistent and physically interpretable simulations of macromolecules in crystals, and allow structural heterogeneity and disorder to be systematically explored on different scales. The new approach is demonstrated on several case studies and the SARS-CoV-2 main protease, and demonstrates how the choice of disorder model affects the type of disorder that is sampled by the restrained molecular-dynamics simulation.

A method for intuitively extracting macromolecular dynamics from structural disorder.

Macromolecular dynamics manifest as disorder in structure determination, which is subsequently accounted for by displacement parameters (also called temperature factors, or B-factors) or alternate conformations. Though B-factors contain detailed information about structural dynamics, they are the total of multiple sources of disorder, making them difficult to interpret and thus little-used in structural analysis. We report here an analytical approach for decomposing molecular disorder into a parsimonious hierarchical series of contributions, providing an intuitive basis for quantitative structural-dynamics analysis. We demonstrate the decomposition of disorder on example SARS-CoV-2 and STEAP4 structures, from both crystallographic and cryo-electron microscopy data, and reveal how understanding of the macromolecular disorder leads to deeper understanding of molecular motions and flexibility, and suggests hypotheses for molecular mechanisms.

C1q binding to surface-bound IgG is stabilized by C1r2s2 proteases.

Complement is an important effector mechanism for antibody-mediated clearance of infections and tumor cells. Upon binding to target cells, the antibody's constant (Fc) domain recruits complement component C1 to initiate a proteolytic cascade that generates lytic pores and stimulates phagocytosis. The C1 complex (C1qr2s2) consists of the large recognition protein C1q and a heterotetramer of proteases C1r and C1s (C1r2s2). While interactions between C1 and IgG-Fc are believed to be mediated by the globular heads of C1q, we here find that C1r2s2 proteases affect the capacity of C1q to form an avid complex with surface-bound IgG molecules (on various 2,4-dinitrophenol [DNP]-coated surfaces and pathogenic Staphylococcus aureus). The extent to which C1r2s2 contributes to C1q-IgG stability strongly differs between human IgG subclasses. Using antibody engineering of monoclonal IgG, we reveal that hexamer-enhancing mutations improve C1q-IgG stability, both in the absence and presence of C1r2s2 In addition, hexamer-enhanced IgGs targeting S. aureus mediate improved complement-dependent phagocytosis by human neutrophils. Altogether, these molecular insights into complement binding to surface-bound IgGs could be important for optimal design of antibody therapies.

Releasing Nonperipheral Subunits from Protein Complexes in the Gas Phase.

The quaternary structure is an important feature regulating protein function. Native mass spectrometry contributes to untangling quaternary structures by preserving the integrity of protein complexes in the gas phase. Tandem mass spectrometry by collision-induced dissociation (CID) can then be used to release subunits from these intact complexes, thereby providing structural information on the stoichiometry and topology. Cumulatively, such studies have revealed the preferred release of peripheral subunits during CID. In contrast, here we describe and focus on dissociation pathways that release nonperipheral subunits from hetero-complexes in CID at high collision energies. We find that nonperipheral subunits are ejected with a high propensity, as a consequence of sequential dissociation events, upon initial removal of peripheral subunits. Alternatively, nonperipheral subunits can be released directly from a charge-reduced or an elongated intact complex. As demonstrated here for a range of protein assemblies, releasing nonperipheral subunits under controlled conditions may provide unique structural information on the stoichiometry and topology of protein complexes.

Implications for tetraspanin-enriched microdomain assembly based on structures of CD9 with EWI-F.

Tetraspanins are eukaryotic membrane proteins that contribute to a variety of signaling processes by organizing partner-receptor molecules in the plasma membrane. How tetraspanins bind and cluster partner receptors into tetraspanin-enriched microdomains is unknown. Here, we present crystal structures of the large extracellular loop of CD9 bound to nanobodies 4C8 and 4E8 and, the cryo-EM structure of 4C8-bound CD9 in complex with its partner EWI-F. CD9-EWI-F displays a tetrameric arrangement with two central EWI-F molecules, dimerized through their ectodomains, and two CD9 molecules, one bound to each EWI-F transmembrane helix through CD9-helices h3 and h4. In the crystal structures, nanobodies 4C8 and 4E8 bind CD9 at loops C and D, which is in agreement with the 4C8 conformation in the CD9-EWI-F complex. The complex varies from nearly twofold symmetric (with the two CD9 copies nearly anti-parallel) to ca. 50° bent arrangements. This flexible arrangement of CD9-EWI-F with potential CD9 homo-dimerization at either end provides a "concatenation model" for forming short linear or circular assemblies, which may explain the occurrence of tetraspanin-enriched microdomains.

An Elegant Four-Helical Fold in NOX and STEAP Enzymes Facilitates Electron Transport across Biomembranes-Similar Vehicle, Different Destination.

The ferric reductase superfamily comprises several oxidoreductases that use an intracellular electron source to reduce an extracellular acceptor substrate. NADPH oxidases (NOXs) and six-transmembrane epithelial antigen of the prostate enzymes (STEAPs) are iconic members of the superfamily. NOXs produce extracellular reactive oxygen species that exert potent bactericidal activities and trigger redox-signaling cascades that regulate cell division and differentiation. STEAPs catalyze the reduction of extracellular iron and copper which is necessary for the bioavailability of these essential elements. Both NOXs and STEAPs are present as multiple isozymes with distinct regulatory properties and physiological roles. Despite the important roles of NOXs and STEAPs in human physiology and despite their wide involvement in diseases like cancer, their mode of action at the molecular level remained incompletely understood for a long time, in part due to the absence of high-resolution models of the complete enzymes. Our two laboratories have elucidated the three-dimensional structures of NOXs and STEAPs, providing key insight into their mechanisms and evolution. The enzymes share a conserved transmembrane helical domain with an eye-catching hourglass shape. On the extracellular side, a heme prosthetic group is at the bottom of a pocket where the substrate (O2 in NOX, chelated iron or copper in STEAP) is reduced. On the intracellular side, the inner heme of NOX and the FAD of STEAP are bound to topological equivalent sites. This is a rare case where critical amino acid substitutions and local conformational changes enable a cofactor (heme vs FAD) swap between two structurally and functionally conserved scaffolds. The catalytic core of these enzymes is completed by distinct cytosolic NADPH-binding domains that are topologically unrelated (a ferredoxin reductase-like flavoprotein domain in NOX and a F420H2:NADP+-like domain in STEAP), feature different quaternary structures, and underlie specific regulatory mechanisms. Despite their differences, these domains all establish electron-transfer chains that direct the electrons from NADPH to the transmembrane domain. The multistep nature of the process and the chemical nature of the products pose considerable problems in the enzymatic assays. We learned that great care must be exerted in the validation of a candidate inhibitor. Multiple orthogonal assays are required to rule out off-target effects such as ROS-scavenging activities or nonspecific interference with the enzyme redox chain. The structural analysis of STEAP/NOX enzymes led us to further notice that their transmembrane heme-binding topology is shared by other enzymes. We found that the core domain of the cytochrome b subunits of the mitochondrial complex III and photosynthetic cytochrome b6f are closely related to NOXs and STEAPs and likely arose from the same ancestor protein. This observation expands the substrate portfolio of the superfamily since cytochromes b act on ubiquinone. The rigidly packed helices of the NOX/STEAP/cytochrome b domain contrast with the more malleable membrane proteins like ion channels or amino-acid transporters, which undergo large conformational changes to allow passage of relatively large metabolites. This notion of a rigid hourglass scaffold found an unexpected confirmation in the observation, revealed by structural comparisons, that an helical bundle identical to the NOX/STEAP/cytochrome b enzymes is featured by a de novo designed heme-binding protein, PS1. Apparently, nature and protein designers have independently converged to this fold as a versatile scaffold for heme-mediated reactions. The challenge is now to uncover the molecular mechanisms that implement the isozyme-specific regulation of the enzyme functions and develop much needed inhibitors and modulators for chemical biology and drug design studies.

Cryo-electron microscopy structure and potential enzymatic function of human six-transmembrane epithelial antigen of the prostate 1 (STEAP1).

Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising therapeutic target to manage these diseases. It shares sequence homology with three enzymes (STEAP2-STEAP4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ∼3.0-Šcryo-EM structure of trimeric human STEAP1 bound to three antigen-binding fragments (Fabs) of the clinically used antibody mAb120.545. The structure revealed that STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells revealed that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, suggesting that STEAP1 functions as a ferric reductase in STEAP heterotrimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and may be useful in the design of new therapeutic strategies to target STEAP1 in cancer.

Site-specific functionality and tryptophan mimicry of lipidation in tetraspanin CD9.

Lipidation of transmembrane proteins regulates many cellular activities, including signal transduction, cell-cell communication, and membrane trafficking. However, how lipidation at different sites in a membrane protein affects structure and function remains elusive. Here, using native mass spectrometry we determined that wild-type human tetraspanins CD9 and CD81 exhibit nonstochastic distributions of bound acyl chains. We revealed CD9 lipidation at its three most frequent lipidated sites suffices for EWI-F binding, while cysteine-to-alanine CD9 mutations markedly reduced binding of EWI-F. EWI-F binding by CD9 was rescued by mutating all or, albeit to a lesser extent, only the three most frequently lipidated sites into tryptophans. These mutations did not affect the nanoscale distribution of CD9 in cell membranes, as shown by super-resolution microscopy using a CD9-specific nanobody. Thus, these data demonstrate site-specific, possibly conformation-dependent, functionality of lipidation in tetraspanin CD9 and identify tryptophan mimicry as a possible biochemical approach to study site-specific transmembrane-protein lipidation.

Author Correction: 'Stealth' corporate innovation: an emerging threat for therapeutic drug development.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Insights Into Enhanced Complement Activation by Structures of Properdin and Its Complex With the C-Terminal Domain of C3b.

Properdin enhances complement-mediated opsonization of targeted cells and particles for immune clearance. Properdin occurs as dimers, trimers and tetramers in human plasma, which recognize C3b-deposited surfaces, promote formation, and prolong the lifetime of C3bBb-enzyme complexes that convert C3 into C3b, thereby enhancing the complement-amplification loop. Here, we report crystal structures of monomerized properdin, which was produced by co-expression of separate N- and C-terminal constructs that yielded monomer-sized properdin complexes that stabilized C3bBb. Consistent with previous low-resolution X-ray and EM data, the crystal structures revealed ring-shaped arrangements that are formed by interactions between thrombospondin type-I repeat (TSR) domains 4 and 6 of one protomer interacting with the N-terminal domain (which adopts a short transforming-growth factor B binding protein-like fold) and domain TSR1 of a second protomer, respectively. Next, a structure of monomerized properdin in complex with the C-terminal domain of C3b showed that properdin-domain TSR5 binds along the C-terminal α-helix of C3b, while two loops, one from domain TSR5 and one from TSR6, extend and fold around the C3b C-terminus like stirrups. This suggests a mechanistic model in which these TSR5 and TSR6 "stirrups" bridge interactions between C3b and factor B or its fragment Bb, and thereby enhance formation of C3bB pro-convertases and stabilize C3bBb convertases. In addition, properdin TSR6 would sterically block binding of the protease factor I to C3b, thus limiting C3b proteolytic degradation. The presence of a valine instead of a third tryptophan in the canonical Trp-ladder of TSR domains in TSR4 allows a remarkable ca. 60°-domain bending motion of TSR4. Together with variable positioning of TSR2 and, putatively, TSR3, this explains the conformational flexibility required for properdin to form dimers, trimers, and tetramers. In conclusion, the results indicate that binding avidity of oligomeric properdin is needed to distinguish surface-deposited C3b molecules from soluble C3b or C3 and suggest that properdin-mediated interactions bridging C3b-B and C3b-Bb enhance affinity, thus promoting convertase formation and stabilization. These mechanisms explain the enhancement of complement-mediated opsonization of targeted cells and particle for immune clearance.

Insights into IgM-mediated complement activation based on in situ structures of IgM-C1-C4b.

Antigen binding by serum Ig-M (IgM) protects against microbial infections and helps to prevent autoimmunity, but causes life-threatening diseases when mistargeted. How antigen-bound IgM activates complement-immune responses remains unclear. We present cryoelectron tomography structures of IgM, C1, and C4b complexes formed on antigen-bearing lipid membranes by normal human serum at 4 °C. The IgM-C1-C4b complexes revealed C4b product release as the temperature-limiting step in complement activation. Both IgM hexamers and pentamers adopted hexagonal, dome-shaped structures with Fab pairs, dimerized by hinge domains, bound to surface antigens that support a platform of Fc regions. C1 binds IgM through widely spread C1q-collagen helices, with C1r proteases pointing outward and C1s bending downward and interacting with surface-attached C4b, which further interacts with the adjacent IgM-Fab2 and globular C1q-recognition unit. Based on these data, we present mechanistic models for antibody-mediated, C1q-transmitted activation of C1 and for C4b deposition, while further conformational rearrangements are required to form C3 convertases.

Anti-LRP5/6 VHHs promote differentiation of Wnt-hypersensitive intestinal stem cells.

Wnt-induced ß-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced ß-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third ß-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors.