The potent HIV-1 capsid inhibitor GS-6207 is an investigational principal component of long-acting antiretroviral therapy. We found that GS-6207 inhibits HIV-1 by stabilizing and thereby preventing ...functional disassembly of the capsid shell in infected cells. X-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that stabilize the curved capsid lattice. In addition, GS-6207 interferes with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. These findings elucidate structural insights into the multimodal, potent antiviral activity of GS-6207 and provide a means for rationally developing second-generation therapies.
Cellular proteins CPSF6, NUP153 and SEC24C play crucial roles in HIV-1 infection. While weak interactions of short phenylalanine-glycine (FG) containing peptides with isolated capsid hexamers have ...been characterized, how these cellular factors functionally engage with biologically relevant mature HIV-1 capsid lattices is unknown. Here we show that prion-like low complexity regions (LCRs) enable avid CPSF6, NUP153 and SEC24C binding to capsid lattices. Structural studies revealed that multivalent CPSF6 assembly is mediated by LCR-LCR interactions, which are templated by binding of CPSF6 FG peptides to a subset of hydrophobic capsid pockets positioned along adjoining hexamers. In infected cells, avid CPSF6 LCR-mediated binding to HIV-1 cores is essential for functional virus-host interactions. The investigational drug lenacapavir accesses unoccupied hydrophobic pockets in the complex to potently impair HIV-1 inside the nucleus without displacing the tightly bound cellular cofactor from virus cores. These results establish previously undescribed mechanisms of virus-host interactions and antiviral action.
Mass spectrometry (MS) is a central analytical technique used to study proteins and biomolecules. It measures mass‐to‐charge ratio of ions to identify and quantify molecules in simple and complex ...mixtures. Technological advancement in instrumentation, sample preparation methodologies, and data analysis workflows continue to push the capabilities of MS to answer more complicated questions and vice versa. Structural proteomics uses MS‐based methodologies to characterize protein structure. Specifically, but not limited to, hydrogen deuterium exchange MS (HDX‐MS) and crosslinking MS (XL‐MS) are complementary techniques that capture the structural plasticity inherent to proteins in solution. This review is intended to present recent progress in HDX‐MS and XL‐MS that have allowed these techniques to be used not only for simple recombinant protein systems but with complex cellular systems.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Among various protein posttranslational modifiers, poly-ADP-ribose polymerase 1 (PARP1) is a key player for regulating numerous cellular processes and events through enzymatic attachments of target ...proteins with ADP-ribose units donated by nicotinamide adenine dinucleotide (NAD
+
). Human PARP1 is involved in the pathogenesis and progression of many diseases. PARP1 inhibitors have received approvals for cancer treatment. Despite these successes, our understanding about PARP1 remains limited, partially due to the presence of various ADP-ribosylation reactions catalyzed by other PARPs and their overlapped cellular functions. Here we report a synthetic NAD
+
featuring an adenosyl 3′-azido substitution. Acting as an ADP-ribose donor with high activity and specificity for human PARP1, this compound enables labelling and profiling of possible protein substrates of endogenous PARP1. It provides a unique and valuable tool for studying PARP1 in biology and pathology and may shed light on the development of PARP isoform-specific modulators.
An analogue of nicotinamide adenine dinucleotide (NAD
+
) featuring an azido group at 3′-OH of adenosine moiety is found to possess high specificity for human PARP1-catalyzed protein poly-ADP-ribosylation.
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IJS, KILJ, NUK, UL, UM, UPUK
Liver receptor homologue-1 (LRH-1) is a phospholipid-sensing nuclear receptor that has shown promise as a target for alleviating intestinal inflammation and metabolic dysregulation in the liver. ...LRH-1 contains a large ligand-binding pocket, but generating synthetic modulators has been challenging. We have had recent success generating potent and efficacious agonists through two distinct strategies. We targeted residues deep within the pocket to enhance compound binding and residues at the mouth of the pocket to mimic interactions made by phospholipids. Here, we unite these two designs into one molecule to synthesize the most potent LRH-1 agonist to date. Through a combination of global transcriptomic, biochemical, and structural studies, we show that selective modulation can be driven through contacting deep versus surface polar regions in the pocket. While deep pocket contacts convey high affinity, contacts with the pocket mouth dominate allostery and provide a phospholipid-like transcriptional response in cultured cells.
Coronavirus (CoV) nonstructural proteins (nsps) assemble to form the replication–transcription complex (RTC) responsible for viral RNA synthesis. nsp7 and nsp8 are important cofactors of the RTC, as ...they interact and regulate the activity of RNA-dependent RNA polymerase and other nsps. To date, no structure of the full-length SARS-CoV-2 nsp7:nsp8 complex has been published. The current understanding of this complex is based on structures from truncated constructs, with missing electron densities, or from related CoV species where SARS-CoV-2 nsp7 and nsp8 share upward of 90% sequence identity. Despite available structures solved using crystallography and cryo-EM representing detailed static snapshots of the nsp7:nsp8 complex, it is evident that the complex has a high degree of structural plasticity. However, relatively little is known about the conformational dynamics of the individual proteins and how they complex to interact with other nsps. Here, the solution-based structural proteomic techniques, hydrogen–deuterium exchange mass spectrometry (HDX-MS) and cross-linking mass spectrometry (XL-MS), illuminate the dynamics of SARS-CoV-2 full-length nsp7 and nsp8 proteins and the nsp7:nsp8 protein complex. Results presented from the two techniques are complementary and validate the interaction surfaces identified from the published three-dimensional heterotetrameric crystal structure of the SARS-CoV-2 truncated nsp7:nsp8 complex. Furthermore, mapping of XL-MS data onto higher-order complexes suggests that SARS-CoV-2 nsp7 and nsp8 do not assemble into a hexadecameric structure as implied by the SARS-CoV full-length nsp7:nsp8 crystal structure. Instead, our results suggest that the nsp7:nsp8 heterotetramer can dissociate into a stable dimeric unit that might bind to nsp12 in the RTC without significantly altering nsp7–nsp8 interactions.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
Protein poly-ADP-ribosylation (PARylation) is a heterogeneous and dynamic post-translational modification regulated by various writers, readers, and erasers. It participates in a variety of ...biological events and is involved in many human diseases. Currently, tools and technologies have yet to be developed for unambiguously defining readers and erasers of individual PARylated proteins or cognate PARylated proteins for known readers and erasers. Here, we report the generation of a bifunctional nicotinamide adenine dinucleotide (NAD+) characterized by diazirine-modified adenine and clickable ribose. By serving as an excellent substrate for poly-ADP-ribose polymerase 1 (PARP1)-catalyzed PARylation, the generated bifunctional NAD+ enables photo-cross-linking and enrichment of PARylation-dependent interacting proteins for proteomic identification. This bifunctional NAD+ provides an important tool for mapping cellular interaction networks centered on protein PARylation, which are essential for elucidating the roles of PARylation-based signals or activities in physiological and pathophysiological processes.
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IJS, KILJ, NUK, PNG, UL, UM, UPUK
BackgroundDespite numerous therapeutic options, safe and curative therapy is unavailable for most patients with chronic lymphocytic leukemia (CLL). A drawback of current therapies such as the ...anti-CD20 monoclonal antibody (mAb) rituximab is the elimination of all healthy B cells, resulting in impaired humoral immunity. We previously reported the identification of a patient-derived, CLL-binding mAb, JML-1, and identified sialic acid-binding immunoglobulin-like lectin-6 (Siglec-6) as the target of JML-1. Although little is known about Siglec-6, it appears to be an attractive target for cancer immunotherapy due to its absence on most healthy cells and tissues.MethodsWe used a target-specific approach to mine for additional patient-derived anti-Siglec-6 mAbs. To assess the therapeutic utility of targeting Siglec-6 in the context of CLL, T cell-recruiting bispecific antibodies (T-biAbs) that bind to Siglec-6 and CD3 were engineered into single-chain variable fragment–Fc and dual-affinity retargeting (DART)–Fc constructs. T-biAbs were evaluated for their activity in vitro, ex vivo, and in vivo.ResultsWe discovered the anti-Siglec-6 mAbs RC-1 and RC-2, which bind with higher affinity than JML-1 yet maintain similar specificity. Both JML-1 and RC-1 T-biAbs were effective at activating T cells and killing Siglec-6+ target cells. The RC-1 clone in the DART–Fc format was the most potent T-biAb tested and was the only anti-Siglec-6 T-biAb that eliminated Siglec-6+ primary CLL cells via autologous T cells at pathological T-to-CLL cell ratios. Tested at healthy T-to-B cell ratios, it also eliminated a Siglec-6+ fraction of primary B cells from healthy donors. The subpicomolar potency of the DART–Fc format was attributed to the reduction in the length and flexibility of the cytolytic synapse. Furthermore, the RC-1 T-biAb was effective at clearing MEC1 CLL cells in vivo and demonstrated a circulatory half-life of over 7 days.ConclusionSiglec-6-targeting T-biAbs are highly potent and specific for eliminating Siglec-6+ leukemic and healthy B cells while sparing Siglec-6− healthy B cells, suggesting a unique treatment strategy for CLL with diminished suppression of humoral immunity. Our data corroborate reports that T-biAb efficacy is dependent on synapse geometry and reveal that synapse architecture can be tuned via antibody engineering. Our fully human anti-Siglec-6 antibodies and T-biAbs have potential for cancer immunotherapy.Trial registration numberNCT00923507.
SARS-CoV-2, a human coronavirus, is the causative agent of the COVID-19 pandemic. Its genome is translated into two large polyproteins subsequently cleaved by viral papain-like protease and main ...protease (Mpro). Polyprotein processing is essential yet incompletely understood. We studied Mpro-mediated processing of the nsp7-11 polyprotein, whose mature products include cofactors of the viral replicase, and identified the order of cleavages. Integrative modeling based on mass spectrometry (including hydrogen-deuterium exchange and cross-linking) and x-ray scattering yielded a nsp7-11 structural ensemble, demonstrating shared secondary structural elements with individual nsps. The pattern of cross-links and HDX footprint of the C145A Mpro and nsp7-11 complex demonstrate preferential binding of the enzyme active site to the polyprotein junction sites and additional transient contacts to help orient the enzyme on its substrate for cleavage. Last, proteolysis assays were used to characterize the effect of inhibitors/binders on Mpro processing/inhibition using the nsp7-11 polyprotein as substrate.
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