The neoepitope of the complement C5b-9 Membrane Attack Complex is formed by proximity of adjacent ancillary regions of C9.

The Membrane Attack Complex (MAC) is responsible for forming large ß-barrel channels in the membranes of pathogens, such as gram-negative bacteria. Off-target MAC assembly on endogenous tissue is associated with inflammatory diseases and cancer. Accordingly, a human C5b-9 specific antibody, aE11, has been developed that detects a neoepitope exposed in C9 when it is incorporated into the C5b-9 complex, but not present in the plasma native C9. For nearly four decades aE11 has been routinely used to study complement, MAC-related inflammation, and pathophysiology. However, the identity of C9 neoepitope remains unknown. Here, we determined the cryo-EM structure of aE11 in complex with polyC9 at 3.2 Å resolution. The aE11 binding site is formed by two separate surfaces of the oligomeric C9 periphery and is therefore a discontinuous quaternary epitope. These surfaces are contributed by portions of the adjacent TSP1, LDLRA, and MACPF domains of two neighbouring C9 protomers. By substituting key antibody interacting residues to the murine orthologue, we validated the unusual binding modality of aE11. Furthermore, aE11 can recognise a partial epitope in purified monomeric C9 in vitro, albeit weakly. Taken together, our results reveal the structural basis for MAC recognition by aE11.

Helical ultrastructure of the metalloprotease meprin α in complex with a small molecule inhibitor.

The zinc-dependent metalloprotease meprin α is predominantly expressed in the brush border membrane of proximal tubules in the kidney and enterocytes in the small intestine and colon. In normal tissue homeostasis meprin α performs key roles in inflammation, immunity, and extracellular matrix remodelling. Dysregulated meprin α is associated with acute kidney injury, sepsis, urinary tract infection, metastatic colorectal carcinoma, and inflammatory bowel disease. Accordingly, meprin α is the target of drug discovery programs. In contrast to meprin ß, meprin α is secreted into the extracellular space, whereupon it oligomerises to form giant assemblies and is the largest extracellular protease identified to date (~6 MDa). Here, using cryo-electron microscopy, we determine the high-resolution structure of the zymogen and mature form of meprin α, as well as the structure of the active form in complex with a prototype small molecule inhibitor and human fetuin-B. Our data reveal that meprin α forms a giant, flexible, left-handed helical assembly of roughly 22 nm in diameter. We find that oligomerisation improves proteolytic and thermal stability but does not impact substrate specificity or enzymatic activity. Furthermore, structural comparison with meprin ß reveal unique features of the active site of meprin α, and helical assembly more broadly.

Structure of the metastatic factor P-Rex1 reveals a two-layered autoinhibitory mechanism.

P-Rex (PI(3,4,5)P3-dependent Rac exchanger) guanine nucleotide exchange factors potently activate Rho GTPases. P-Rex guanine nucleotide exchange factors are autoinhibited, synergistically activated by Gßγ and PI(3,4,5)P3 binding and dysregulated in cancer. Here, we use X-ray crystallography, cryogenic electron microscopy and crosslinking mass spectrometry to determine the structural basis of human P-Rex1 autoinhibition. P-Rex1 has a bipartite structure of N- and C-terminal modules connected by a C-terminal four-helix bundle that binds the N-terminal Pleckstrin homology (PH) domain. In the N-terminal module, the Dbl homology (DH) domain catalytic surface is occluded by the compact arrangement of the DH-PH-DEP1 domains. Structural analysis reveals a remarkable conformational transition to release autoinhibition, requiring a 126° opening of the DH domain hinge helix. The off-axis position of Gßγ and PI(3,4,5)P3 binding sites further suggests a counter-rotation of the P-Rex1 halves by 90° facilitates PH domain uncoupling from the four-helix bundle, releasing the autoinhibited DH domain to drive Rho GTPase signaling.

Thermoresponsive chiral plasmonic nanoparticles.

Chiral metallic nanoparticles can exhibit novel plasmonic circular dichroism (PCD) in the ultraviolet and visible range of the electromagnetic spectrum. Here, we investigate how thermoresponsive dielectric nanoenvironments will influence such PCD responses through poly(N-isopropylacrylamide) (PNIPAM) modified chiral gold nanorods (AuNRs). We observed the temperature-dependent chiral plasmonic responses distinctly from unmodified counterparts. As for the modified systems, the PCD peaks for both L-AuNRs and D-AuNRs at 50 °C red shifted simultaneously with enhanced intensities compared to the results at 20 °C. In contrast, the unmodified L-AuNRs and D-AuNRs exhibited no peak shift with reduced intensities. Subsequent simulation and experimental studies demonstrated that the enhanced PCD was attributed to PNIPAM chain collapse causing the increase of the refractive index by expelling minute water out of the corona surrounding chiral plasmonic AuNRs. Notably, such thermoresponsive chiral plasmonic responses are reversible, general, and extendable to other types of chiral plasmonic nanoparticles.

The cryo-EM structure of the human neurofibromin dimer reveals the molecular basis for neurofibromatosis type 1.

Neurofibromin (NF1) mutations cause neurofibromatosis type 1 and drive numerous cancers, including breast and brain tumors. NF1 inhibits cellular proliferation through its guanosine triphosphatase-activating protein (GAP) activity against rat sarcoma (RAS). In the present study, cryo-electron microscope studies reveal that the human ~640-kDa NF1 homodimer features a gigantic 30 × 10 nm array of α-helices that form a core lemniscate-shaped scaffold. Three-dimensional variability analysis captured the catalytic GAP-related domain and lipid-binding SEC-PH domains positioned against the core scaffold in a closed, autoinhibited conformation. We postulate that interaction with the plasma membrane may release the closed conformation to promote RAS inactivation. Our structural data further allow us to map the location of disease-associated NF1 variants and provide a long-sought-after structural explanation for the extreme susceptibility of the molecule to loss-of-function mutations. Collectively these findings present potential new routes for therapeutic modulation of the RAS pathway.

Structural analysis of the PTEN:P-Rex2 signaling complex reveals how cancer-associated mutations coordinate to hyperactivate Rac1.

The dual-specificity phosphatase PTEN functions as a tumor suppressor by hydrolyzing PI(3,4,5)P3 to PI(4,5)P2 to inhibit PI3K-AKT signaling and cellular proliferation. P-Rex2 is a guanine nucleotide exchange factor for Rho GTPases and can be activated by Gßγ subunits downstream of G protein-coupled receptor signaling and by PI(3,4,5)P3 downstream of receptor tyrosine kinases. The PTEN:P-Rex2 complex is a commonly mutated signaling node in metastatic cancer. Assembly of the PTEN:P-Rex2 complex inhibits the activity of both proteins, and its dysregulation can drive PI3K-AKT signaling and cellular proliferation. Here, using cross-linking mass spectrometry and functional studies, we gained mechanistic insights into PTEN:P-Rex2 complex assembly and coinhibition. We found that PTEN was anchored to P-Rex2 by interactions between the PDZ-interacting motif in the PTEN C-terminal tail and the second PDZ domain of P-Rex2. This interaction bridged PTEN across the P-Rex2 surface, preventing PI(3,4,5)P3 hydrolysis. Conversely, PTEN both allosterically promoted an autoinhibited conformation of P-Rex2 and blocked its binding to Gßγ. In addition, we observed that the PTEN-deactivating mutations and P-Rex2 truncations combined to drive Rac1 activation to a greater extent than did either single variant alone. These insights enabled us to propose a class of gain-of-function, cancer-associated mutations within the PTEN:P-Rex2 interface that uncouple PTEN from the inhibition of Rac1 signaling.

Amyloidogenicity at a Distance: How Distal Protein Regions Modulate Aggregation in Disease.

The misfolding of proteins to form amyloid is a key pathological feature of several progressive, and currently incurable, diseases. A mechanistic understanding of the pathway from soluble, native protein to insoluble amyloid is crucial for therapeutic design, and recent efforts have helped to elucidate the key molecular events that trigger protein misfolding. Generally, either global or local structural perturbations occur early in amyloidogenesis to expose aggregation-prone regions of the protein that can then self-associate to form toxic oligomers. Surprisingly, these initiating structural changes are often caused or influenced by protein regions distal to the classically amyloidogenic sequences. Understanding the importance of these distal regions in the pathogenic process has highlighted many remaining knowledge gaps regarding the precise molecular events that occur in classic aggregation pathways. In this review, we discuss how these distal regions can influence aggregation in disease and the recent technical and conceptual advances that have allowed this insight.

Enhanced molecular mobility of ordinarily structured regions drives polyglutamine disease.

Polyglutamine expansion is a hallmark of nine neurodegenerative diseases, with protein aggregation intrinsically linked to disease progression. Although polyglutamine expansion accelerates protein aggregation, the misfolding process is frequently instigated by flanking domains. For example, polyglutamine expansion in ataxin-3 allosterically triggers the aggregation of the catalytic Josephin domain. The molecular mechanism that underpins this allosteric aggregation trigger remains to be determined. Here, we establish that polyglutamine expansion increases the molecular mobility of two juxtaposed helices critical to ataxin-3 deubiquitinase activity. Within one of these helices, we identified a highly amyloidogenic sequence motif that instigates aggregation and forms the core of the growing fibril. Critically, by mutating residues within this key region, we decrease local structural fluctuations to slow ataxin-3 aggregation. This provides significant insight, down to the molecular level, into how polyglutamine expansion drives aggregation and explains the positive correlation between polyglutamine tract length, protein aggregation, and disease severity.