Molecular basis for GIGYF-TNRC6 complex assembly.

The GIGYF proteins interact with 4EHP and RNA-associated proteins to elicit transcript-specific translational repression. However, the mechanism by which the GIGYF1/2-4EHP complex is recruited to its target transcripts remain unclear. Here, we report the crystal structures of the GYF domains from GIGYF1 and GIGYF2 in complex with proline-rich sequences from the miRISC-binding proteins TNRC6C and TNRC6A, respectively. The TNRC6 proline-rich motifs bind to a conserved array of aromatic residues on the surface of the GIGYF1/2 GYF domains, thereby bridging 4EHP to Argonaute-miRNA complexes. Our structures also reveal a phenylalanine residue conserved from yeast to human GYF domains that contributes to GIGYF2 thermostability. The molecular details we outline here are likely to be conserved between GIGYF1/2 and other RNA-binding proteins to elicit 4EHP-mediated repression in different biological contexts.

Structure of the PAN3 pseudokinase reveals the basis for interactions with the PAN2 deadenylase and the GW182 proteins.

The PAN2-PAN3 deadenylase complex functions in general and miRNA-mediated mRNA degradation and is specifically recruited to miRNA targets by GW182/TNRC6 proteins. We describe the PAN3 adaptor protein crystal structure that, unexpectedly, forms intertwined and asymmetric homodimers. Dimerization is mediated by a coiled coil that links an N-terminal pseudokinase to a C-terminal knob domain. The PAN3 pseudokinase binds ATP, and this function is required for mRNA degradation in vivo. We further identified conserved surfaces required for mRNA degradation, including the binding surface for the PAN2 deadenylase on the knob domain. The most remarkable structural feature is the presence of a tryptophan-binding pocket at the dimer interface, which mediates binding to TNRC6C in human cells. Together, our data reveal the structural basis for the interaction of PAN3 with PAN2 and the recruitment of the PAN2-PAN3 complex to miRNA targets by TNRC6 proteins.

Structural reconstruction of protein ancestry.

Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B- and T-cell receptors), heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen. High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence of phylogenetic evidence.

Human Antibody Bispecifics through Phage Display Selection.

We developed a repertoire approach to generate human antibody bispecifics. Using phage display selection of antibody heavy chains in the presence of a competitor light chain and providing a cognate light chain with an affinity handle, we identified mutations that prevent heavy/light chain mispairing. The strategy allows for the selection of human antibody chains that autonomously assemble into bispecifics.

Cryo-EM analysis of a domain antibody bound rotary ATPase complex.

The bacterial A/V-type ATPase/synthase rotary motor couples ATP hydrolysis/synthesis with proton translocation across biological membranes. The A/V-type ATPase/synthase from Thermus thermophilus has been extensively studied both structurally and functionally for many years. Here we provide an 8.7Å resolution cryo-electron microscopy 3D reconstruction of this complex bound to single-domain antibody fragments, small monomeric antibodies containing just the variable heavy domain. Docking of known structures into the density revealed the molecular orientation of the domain antibodies, suggesting that structure determination of co-domain antibody:protein complexes could be a useful avenue for unstable or smaller proteins. Although previous studies suggested that the presence of fluoroaluminate in this complex could change the rotary state of this enzyme, we observed no gross structural rearrangements under these conditions.

Fully Human VH Single Domains That Rival the Stability and Cleft Recognition of Camelid Antibodies.

Human VH single domains represent a promising class of antibody fragments with applications as therapeutic modalities. Unfortunately, isolated human VH domains also generally display poor biophysical properties and a propensity to aggregate. This has encouraged the development of non-human antibody domains as alternative means of antigen recognition and, in particular, camelid (VHH) domains. Naturally devoid of light chain partners, these domains are characterized by favorable biophysical properties and propensity for cleft binding, a highly desirable characteristic, allowing the targeting of cryptic epitopes. In contrast, previously reported structures of human VH single domains had failed to recapitulate this property. Here we report the engineering and characterization of phage display libraries of stable human VH domains and the selection of binders against a diverse set of antigens. Unlike "camelized" human domains, the domains do not rely on potentially immunogenic framework mutations and maintain the structure of the VH/VL interface. Structure determination in complex with hen egg white lysozyme revealed an extended VH binding interface, with complementarity-determining region 3 deeply penetrating into the active site cleft, highly reminiscent of what has been observed for camelid domains. Taken together, our results demonstrate that fully human VH domains can be constructed that are not only stable and well expressed but also rival the cleft binding properties of camelid antibodies.

Immunocytokines with target cell-restricted IL-15 activity for treatment of B cell malignancies.

Despite the advances in cancer treatment achieved, for example, by the CD20 antibody rituximab, an urgent medical need remains to optimize the capacity of such antibodies to induce antibody-dependent cellular cytotoxicity (ADCC) that determines therapeutic efficacy. The cytokine IL-15 stimulates proliferation, activation, and cytolytic capacity of NK cells, but broad clinical use is prevented by short half-life, poor accumulation at the tumor site, and severe toxicity due to unspecific immune activation. We here report modified immunocytokines consisting of Fc-optimized CD19 and CD20 antibodies fused to an IL-15 moiety comprising an L45E-E46K double mutation (MIC+ format). The E46K mutation abrogated binding to IL-15Rα, thereby enabling substitution of physiological trans-presentation by target binding and thus conditional IL-15Rßγ stimulation, whereas the L45E mutation optimized IL-15Rßγ agonism and producibility. In vitro analysis of NK activation, anti-leukemia reactivity, and toxicity using autologous and allogeneic B cells confirmed target-dependent function of MIC+ constructs. Compared with Fc-optimized CD19 and CD20 antibodies, MIC+ constructs mediated superior target cell killing and NK cell proliferation. Mouse models using luciferase-expressing human NALM-6 lymphoma cells, patient acute lymphoblastic leukemia (ALL) cells, and murine EL-4 lymphoma cells transduced with human CD19/CD20 as targets and human and murine NK cells as effectors, respectively, confirmed superior and target-dependent anti-leukemic activity. In summary, MIC+ constructs combine the benefits of Fc-optimized antibodies and IL-15 cytokine activity and mediate superior NK cell immunity with potentially reduced side effects. They thus constitute a promising new immunotherapeutic approach shown here for B cell malignancies.

Discovery of High Affinity Cyclic Peptide Ligands for Human ACE2 with SARS-CoV-2 Entry Inhibitory Activity.

The development of effective antiviral compounds is essential for mitigating the effects of the COVID-19 pandemic. Entry of SARS-CoV-2 virions into host cells is mediated by the interaction between the viral spike (S) protein and membrane-bound angiotensin-converting enzyme 2 (ACE2) on the surface of epithelial cells. Inhibition of this viral protein-host protein interaction is an attractive avenue for the development of antiviral molecules with numerous spike-binding molecules generated to date. Herein, we describe an alternative approach to inhibit the spike-ACE2 interaction by targeting the spike-binding interface of human ACE2 via mRNA display. Two consecutive display selections were performed to direct cyclic peptide ligand binding toward the spike binding interface of ACE2. Through this process, potent cyclic peptide binders of human ACE2 (with affinities in the picomolar to nanomolar range) were identified, two of which neutralized SARS-CoV-2 entry. This work demonstrates the potential of targeting ACE2 for the generation of anti-SARS-CoV-2 therapeutics as well as broad spectrum antivirals for the treatment of SARS-like betacoronavirus infection.

eIF4E-homologous protein (4EHP): a multifarious cap-binding protein.

The cap-binding protein 4EHP/eIF4E2 has been a recent object of interest in the field of post-transcriptional gene regulation and translational control. From ribosome-associated quality control, to RNA decay and microRNA-mediated gene silencing, this member of the eIF4E protein family regulates gene expression through numerous pathways. Low in abundance but ubiquitously expressed, 4EHP interacts with different binding partners to form multiple protein complexes that regulate translation in a variety of biological contexts. Documented functions of 4EHP primarily relate to its role as a translational repressor, but recent findings indicate that it might also participate in the activation of translation in specific settings. In this review, we discuss the known functions, properties and mechanisms that involve 4EHP in the control of gene expression. We also discuss our current understanding of how 4EHP processes are regulated in eukaryotic cells, and the diseases implicated with dysregulation of 4EHP-mediated translational control.

Discovery and characterization of cyclic peptides selective for the C-terminal bromodomains of BET family proteins.

DNA-encoded cyclic peptide libraries can yield high-potency, high-specificity ligands against target proteins. We used such a library to seek ligands that could distinguish between paralogous bromodomains from the closely related bromodomain and extra-terminal domain family of epigenetic regulators. Several peptides isolated from a screen against the C-terminal bromodomain of BRD2, together with new peptides discovered in previous screens against the corresponding domain from BRD3 and BRD4, bound their targets with nanomolar and sub-nanomolar affinities. X-ray crystal structures of several of these bromodomain-peptide complexes reveal diverse structures and binding modes, which nevertheless display several conserved features. Some peptides demonstrate significant paralog-level specificity, although the physicochemical explanations for this specificity are often not clear. Our data demonstrate the power of cyclic peptides to discriminate between very similar proteins with high potency and hint that differences in conformational dynamics might modulate the affinity of these domains for particular ligands.

Novel ACE2 fusion protein with adapting activity against SARS-CoV-2 variants in vitro.

Despite the successful development of vaccines and neutralizing antibodies to limit the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerging variants prolong the pandemic and emphasize the persistent need to develop effective antiviral treatment regimens. Recombinant antibodies directed to the original SARS-CoV-2 have been successfully used to treat established viral disease. However, emerging viral variants escape the recognition by those antibodies. Here we report the engineering of an optimized ACE2 fusion protein, designated ACE2-M, which comprises a human IgG1 Fc domain with abrogated Fc-receptor binding linked to a catalytically-inactive ACE2 extracellular domain that displays increased apparent affinity to the B.1 spike protein. The affinity and neutralization capacity of ACE2-M is unaffected or even enhanced by mutations present in the spike protein of viral variants. In contrast, a recombinant neutralizing reference antibody, as well as antibodies present in the sera of vaccinated individuals, lose activity against such variants. With its potential to resist viral immune escape ACE2-M appears to be particularly valuable in the context of pandemic preparedness towards newly emerging coronaviruses.

Raman spectroscopy system for real-time diagnosis of clinically significant prostate cancer tissue.

Prostate cancer (PCa) is a significant healthcare problem worldwide. Current diagnosis and treatment methods are limited by a lack of precise in vivo tissue analysis methods. Real-time cancer identification and grading could dramatically improve current protocols. Here, we report the testing of a thin optical probe using Raman spectroscopy (RS) and classification methods to detect and grade PCa accurately in real-time. We present the first clinical trial on fresh ex vivo biopsy cores from an 84 patient cohort. Findings from 2395 spectra measured on 599 biopsy cores show high accuracy for diagnosing and grading PCa. We can detect clinically significant PCa from benign and clinically insignificant PCa with 90% sensitivity and 80.2% specificity. We also demonstrate the ability to differentiate cancer grades with 90% sensitivity and specificity ≥82.8%. This work demonstrates the utility of RS for real-time PCa detection and grading during routine transrectal biopsy appointments.

Ethnic disparities in Phyllodes Tumour in Aotearoa New Zealand: a retrospective review.

BACKGROUND: Phyllodes tumour (PT) is a rare breast neoplasm and little is known about its epidemiological risk factors. The literature suggests a higher incidence of PT in Asian patients and other minority ethnic groups. The purpose of this study was to identify whether there was a difference in incidence, grade and age at presentation for patients with PT among minority ethnic groups when compared with European patients in Aotearoa New Zealand (AoNZ). METHODS: A retrospective review was conducted across the three District Health Boards (DHBs) in Auckland, Aotearoa New Zealand (AoNZ), from 2008 to 2018 to investigate the relationship between ethnicity and clinical characteristics of PT. Demographic information and histology reports were reviewed to obtain relevant data. The primary outcome measure was ethnicity and the secondary outcome measures were age at presentation, tumour volume and grade. RESULTS: One hundred and fifty-nine patients were included. The total number of non-European patients were 108 (68%). Minority ethnic groups including Pasifika, Maori and MELAA were overrepresented in the patient cohort. Larger tumour volume was significantly correlated with higher tumour grade (p < 0.001). Pasifika patients presented with larger tumours (p 0.05) and at a younger age (p 0.027) when compared with European patients. CONCLUSION: This study found that patients with PT in AoNZ were significantly overrepresented in Asian, Pasifika and MELAA ethnic groups. Pasifika patients may be at an increased risk of presenting at a younger age with larger, higher grade tumours when compared with European patients. Further research is required to investigate the reasons behind these findings in minority ethnic groups.

Discovery of Cyclic Peptide Ligands to the SARS-CoV-2 Spike Protein Using mRNA Display.

The COVID-19 pandemic, caused by SARS-CoV-2, has led to substantial morbidity, mortality, and disruption globally. Cellular entry of SARS-CoV-2 is mediated by the viral spike protein, and affinity ligands to this surface protein have the potential for applications as antivirals and diagnostic reagents. Here, we describe the affinity selection of cyclic peptide ligands to the SARS-CoV-2 spike protein receptor binding domain (RBD) from three distinct libraries (in excess of a trillion molecules each) by mRNA display. We identified six high affinity molecules with dissociation constants (K D) in the nanomolar range (15-550 nM) to the RBD. The highest affinity ligand could be used as an affinity reagent to detect the spike protein in solution by ELISA, and the cocrystal structure of this molecule bound to the RBD demonstrated that it binds to a cryptic binding site, displacing a ß-strand near the C-terminus. Our findings provide key mechanistic insight into the binding of peptide ligands to the SARS-CoV-2 spike RBD, and the ligands discovered in this work may find future use as reagents for diagnostic applications.

Cryo-EM reveals distinct conformations of E. coli ATP synthase on exposure to ATP.

ATP synthase produces the majority of cellular energy in most cells. We have previously reported cryo-EM maps of autoinhibited E. coli ATP synthase imaged without addition of nucleotide (Sobti et al. 2016), indicating that the subunit ε engages the α, ß and γ subunits to lock the enzyme and prevent functional rotation. Here we present multiple cryo-EM reconstructions of the enzyme frozen after the addition of MgATP to identify the changes that occur when this ε inhibition is removed. The maps generated show that, after exposure to MgATP, E. coli ATP synthase adopts a different conformation with a catalytic subunit changing conformation substantially and the ε C-terminal domain transitioning via an intermediate 'half-up' state to a condensed 'down' state. This work provides direct evidence for unique conformational states that occur in E. coli ATP synthase when ATP binding prevents the ε C-terminal domain from entering the inhibitory 'up' state.

Reducing Inappropriate Lumbar Spine MRI for Low Back Pain: Radiology Support, Communication and Alignment Network.

PURPOSE: The aim of this study is to evaluate the impact of educational sessions on reducing lumbar spine MRI inappropriateness for uncomplicated low back pain and to present our institutional experience on the use of ACR's Radiology Support, Communication and Alignment Network (R-SCAN) program toward achieving appropriateness. METHODS: The R-SCAN web portal was accessed to register a project. Using order entry data, the number of lumbar spine MRI orders placed per month at three family medicine clinics was assessed over a 10-month period. After educational presentations were given at those three clinics highlighting the American College of Physicians and Choosing Wisely campaign imaging guidelines, the number of MRI orders placed was reassessed over an additional 10 months. For a subset of these exams, the ACR Appropriateness Criteria rating of the lumbar spine MRIs were compared between the pre- and posteducation periods. A P value < .05 was considered statistically significant. RESULTS: The average number of monthly MRIs ordered from all three clinics combined was 6.3 during the posteducation period, which was significantly less than during the pre-education period of 10.0 (P = .009). The combined average ACR Appropriateness Criteria rating made at all three clinics was 5.8 after educational sessions, which was significantly higher than the rating of 4.7 before educational sessions (P = .014). CONCLUSION: Clinician education, facilitated by R-SCAN, resulted in a reduced number of MRI lumbar spine studies performed for uncomplicated low back pain and improved appropriateness of those studies as measured by the ACR Appropriateness Criteria rating.

Structural characterisation of TNRC6A nuclear localisation signal in complex with importin-alpha.

The GW182/TNRC6 family of proteins are central scaffolds that link microRNA-associated Argonaute proteins to the cytoplasmic decay machinery for targeted mRNA degradation processes. Although nuclear roles for the GW182/TNRC6 proteins are unknown, recent reports have demonstrated nucleocytoplasmic shuttling activity that utilises the importin-α and importin-ß transport receptors for nuclear translocation. Here we describe the structure of mouse importin-α in complex with the TNRC6A nuclear localisation signal peptide. We further show that the interactions observed between TNRC6A and importin-α are conserved between mouse and human complexes. Our results highlight the ability of monopartite cNLS sequences to maximise contacts at the importin-α major binding site, as well as regions outside the main binding cavities.

Structural basis of antigen recognition: crystal structure of duck egg lysozyme.

Duck egg lysozyme (DEL) is a widely used model antigen owing to its capacity to bind with differential affinity to anti-chicken egg lysozyme antibodies. However, no structures of DEL have so far been reported, and the situation had been complicated by the presence of multiple isoforms and conflicting reports of primary sequence. Here, the structures of two DEL isoforms from the eggs of the commonly used Pekin duck (Anas platyrhynchos) are reported. Using structural analyses in combination with mass spectrometry, non-ambiguous DEL primary sequences are reported. Furthermore, the structures and sequences determined here enable rationalization of the binding affinity of DEL for well documented landmark anti-lysozyme antibodies.

Structural Biology and Regulation of Protein Import into the Nucleus.

Proteins are translated in the cytoplasm, but many need to access the nucleus to perform their functions. Understanding how these nuclear proteins are transported through the nuclear envelope and how the import processes are regulated is therefore an important aspect of understanding cell function. Structural biology has played a key role in understanding the molecular events during the transport processes and their regulation, including the recognition of nuclear targeting signals by the corresponding receptors. Here, we review the structural basis of the principal nuclear import pathways and the molecular basis of their regulation. The pathways involve transport factors that are members of the ß-karyopherin family, which can bind cargo directly (e.g., importin-ß, transportin-1, transportin-3, importin-13) or through adaptor proteins (e.g., importin-α, snurportin-1, symportin-1), as well as unrelated transport factors such as Hikeshi, involved in the transport of heat-shock proteins, and NTF2, involved in the transport of RanGDP. Solenoid proteins feature prominently in these pathways. Nuclear transport factors recognize nuclear targeting signals on the cargo proteins, including the classical nuclear localization signals, recognized by the adaptor importin-α, and the PY nuclear localization signals, recognized by transportin-1. Post-translational modifications, particularly phosphorylation, constitute key regulatory mechanisms operating in these pathways.

Solution structure of a soluble fragment derived from a membrane protein by shotgun proteolysis.

We have previously reported a phage display method for the identification of protein domains on a genome-wide scale (shotgun proteolysis). Here we present the solution structure of a fragment of the Escherichia coli membrane protein yrfF, as identified by shotgun proteolysis, and determined by NMR spectroscopy. Despite the absence of computational predictions, the fragment formed a well-defined beta-barrel structure, distantly falling within the OB-fold classification. Our results highlight the potential of high-throughput experimental approaches for the identification of protein domains for structural studies.