The innate immune response to viruses is critical for the correct establishment of protective ada... more The innate immune response to viruses is critical for the correct establishment of protective adaptive immunity. Amongst the many pathways involved, the NLRP3 [nucleotide-binding oligomerisation domain (NOD)-like receptor protein 3 (NLRP3)] inflammasome has received considerable attention, particularly in the context of immunity and pathogenesis during infection with influenza A (IAV) and SARS-CoV-2, the causative agent of COVID-19. Activation of the NLRP3 inflammasome results in the secretion of the proinflammatory cytokines IL-1β and IL-18, commonly coupled with pyroptotic cell death. While this mechanism is protective and key to host defense, aberrant NLRP3 inflammasome activation causes a hyperinflammatory response and excessive release of cytokines, both locally and systemically. Here, we discuss key molecules in the NLRP3 pathway that have also been shown to have significant roles in innate and adaptive immunity to viruses, including DEAD box helicase X-linked (DDX3X), vimenti...
RIG-I (retinoic-acid inducible gene I, also known as DDX58) is the best characterized receptor wi... more RIG-I (retinoic-acid inducible gene I, also known as DDX58) is the best characterized receptor within the RIG-I like receptor (RLR) family. Together with MDA5 (melanoma differentiation-associated 5) and LGP2 (laboratory of genetics and physiology 2), this family of cytoplasmic pattern recognition receptors (PRRs) are sentinels for intracellular viral RNA that is a product of viral infection. The RLR receptors provide frontline defence against viral infections in most tissues. RLR ligands The RIG-I receptor prefers to bind short (<2000 bp) single-or double-stranded RNA carrying an uncapped 5' triphosphate and additional motifs such as poly-uridine rich RNA motifs. [1][2] RIG-I triggers an immune response to RNA viruses from various families including the paramyxoviruses (e.g. measles), rhabdoviruses (e.g. vesicular stomatitis virus) and orthomyxoviruses (e.g. influenza A). [3][4][5][6][7] MDA5 ligands are poorly characterized, but the preference is for long double-stranded RNA (>2000 bp), such as the replicative form of picornavirus RNA that is found in picornavirus-infected cells. [8][9] LGP2 binds to blunt-ended double-stranded RNA of variable length, [10][11] and also to RNA-bound MDA5 to regulate filament formation. [12] The latter is linked to LGP2's recognition of picornaviruses (e.g. encephalomyocarditis virus), as per MDA5. [13]
Uncontrolled cell proliferation and genomic instability are common features of cancer and can ari... more Uncontrolled cell proliferation and genomic instability are common features of cancer and can arise from, respectively, the loss of cell-cycle control and defective checkpoints. Ubiquitin-mediated proteolysis, ultimately executed by ubiquitin-ligating enzymes (E3s), plays a key part in cell-cycle regulation and is dominated by two multisubunit E3s, the anaphase-promoting complex (or cyclosome) (APC/C) and SKP1–cullin-1–F-box (SCF) complex. We highlight the role of APC/C and the SCF bound to F-box proteins, FBXW7, SKP2, and β-TrCP, in regulating the abundance of select fundamental proteins, primarily during the cell cycle, that are associated with human cancer. The clinical success of the first proteasome inhibitor, bortezomib, in treating multiple myeloma and mantle-cell lymphoma set the precedent for viewing the ubiquitin–proteasome system as a druggable target for cancer. Given that there are more E3s than kinases, selective, small-molecule E3 inhibitors have the potential of open...
Dynamics plays an important but underappreciated role in the interaction between the T cell recep... more Dynamics plays an important but underappreciated role in the interaction between the T cell receptor (TCR) and peptide-bound major histocompatibility complex (pMHC). Crystallographic studies have provided key molecular insights into this interaction; however, due to inherent features of the structural approach, the image of TCR-pMHC interactions that has emerged is a static one. In this review, we discuss how molecular dynamics (MD) simulations can complement and extend current experimental methods aimed at examining TCR-pMHC dynamics. We review the insights obtained from studies that leverage MD approaches, and propose that an integrative strategy that harnesses both MD simulations and structural and biophysical methods will provide new inroads into understanding the transitory and dynamic molecular events that dictate TCR signaling and T cell activation.
Viral disease is one of the greatest burdens for human health worldwide, with an urgent need for ... more Viral disease is one of the greatest burdens for human health worldwide, with an urgent need for efficacious antiviral strategies. While antiviral drugs are available, in many cases, they are prone to the development of drug resistance. A way to overcome drug resistance associated with common antiviral therapies is to develop antivirals targeting host cellular co-factors critical to viral replication, such as DEAD-box helicase 3 X-linked (DDX3X), which plays key roles in RNA metabolism and the antiviral response. Here, we use biochemical/biophysical approaches and infectious assays to show for the first time that the small molecule RK-33 has broad-spectrum antiviral action by inhibiting the enzymatic activities of DDX3X. Importantly, we show that RK-33 is efficacious at low micromolar concentrations in limiting infection by human parainfluenza virus type 3 (hPIV-3), respiratory syncytial virus (RSV), dengue virus (DENV), Zika virus (ZIKV) or West Nile virus (WNV)—for all of which, n...
The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon ... more The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon (IFN) production and antiviral gene expression in response to RNA virus infection. Consequently, RLR signalling is tightly regulated by both host and viral factors. Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade, playing both ubiquitination-dependent and -independent roles in RIG-I-mediated IFN induction. However, additional regulatory roles are emerging. Here, we show a novel interaction between TRIM25 and another protein in the RLR pathway that is essential for type I IFN induction, DEAD-box helicase 3X (DDX3X). In vitro assays and knockdown studies reveal that TRIM25 ubiquitinates DDX3X at lysine 55 (K55) and that TRIM25 and DDX3X cooperatively enhance IFNB1 induction following RIG-I activation, but the latter is independent of TRIM25’s catalytic activity. Furthermore, we found that the influenza A virus no...
The transport of host proteins into and out of the nucleus is key to host function. However, nucl... more The transport of host proteins into and out of the nucleus is key to host function. However, nuclear transport is restricted by nuclear pores that perforate the nuclear envelope. Protein intrinsic disorder is an inherent feature of this selective transport barrier and is also a feature of the nuclear transport receptors that facilitate the active nuclear transport of cargo, and the nuclear transport signals on the cargo itself. Furthermore, intrinsic disorder is an inherent feature of viral proteins and viral strategies to disrupt host nucleocytoplasmic transport to benefit their replication. In this review, we highlight the role that intrinsic disorder plays in the nuclear transport of host and viral proteins. We also describe viral subversion mechanisms of the host nuclear transport machinery in which intrinsic disorder is a feature. Finally, we discuss nuclear import and export as therapeutic targets for viral infectious disease.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DEAD-box helicase 3, X-linked (DDX3X) regulates the retinoic acid-inducible gene I (RIG-I)-like r... more DEAD-box helicase 3, X-linked (DDX3X) regulates the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated antiviral response, but can also be a host factor contributing to the replication of viruses of significance to human health, such as human immunodeficiency virus type 1 (HIV-1). These roles are mediated in part through its ability to actively shuttle between the nucleus and the cytoplasm to modulate gene expression, although the trafficking mechanisms, and impact thereof on immune signaling and viral infection, are incompletely defined. We confirm that DDX3X nuclear export is mediated by the nuclear transporter exportin-1/CRM1, dependent on an N-terminal, leucine-rich nuclear export signal (NES) and the monomeric guanine nucleotide binding protein Ran in activated GTP-bound form. Transcriptome profiling and ELISA show that exportin-1-dependent export of DDX3X to the cytoplasm strongly impacts IFN-β production and the upregulation of immune genes in response to inf...
A structural characterization of the interaction between T cell receptors (TCR) and cognate pepti... more A structural characterization of the interaction between T cell receptors (TCR) and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the X-ray data. We found that in a large number of systems ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship be...
Dengue virus (DENV) threatens almost 70% of the world’s population, with no effective vaccine or ... more Dengue virus (DENV) threatens almost 70% of the world’s population, with no effective vaccine or therapeutic currently available. A key contributor to infection is nuclear localisation in the infected cell of DENV nonstructural protein 5 (NS5) through the action of the host importin (IMP) α/β1 proteins. Here, we used a range of microscopic, virological and biochemical/biophysical approaches to show for the first time that the small molecule GW5074 has anti-DENV action through its novel ability to inhibit NS5–IMPα/β1 interaction in vitro as well as NS5 nuclear localisation in infected cells. Strikingly, GW5074 not only inhibits IMPα binding to IMPβ1, but can dissociate preformed IMPα/β1 heterodimer, through targeting the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity, as shown using analytical ultracentrifugation, thermostability analysis and circular dichroism measurements. Importantly, GW5074 has strong antiviral activity at low µM concentrations...
A structural characterization of the interaction between ab TCRs and cognate peptide-MHC (pMHC) i... more A structural characterization of the interaction between ab TCRs and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell-mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the x-ray data. Focusing on a subset of human pMHC class I systems, we found that in many cases, ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and the discrimination between induced fit and conformational selection models of TCR binding. We show that the currently widespread practice of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from x-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data but also provides previously missing information concerning the dynamics of existing characterized TCR-pMHC interactions.
Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication ... more Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication and decrease the risk of developing acquired immune deficiency syndrome (AIDS). However, its therapeutic value is diminished by the fact that it is associated with drug hypersensitivity reactions in up to 8% of treated patients. This hypersensitivity is strongly associated with patients carrying human leukocyte antigen (HLA)-B*57:01, but not patients carrying closely related alleles. Abacavir’s specificity to HLA-B*57:01 is attributed to its binding site within the peptide-binding cleft and subsequent influence of the repertoire of peptides that can bind HLA-B*57:01. To further our understanding of abacavir-induced hypersensitivity we used molecular dynamics (MD) to analyze the dynamics of three different peptides bound to HLA-B*57:01 in the presence and absence of abacavir or abacavir analogues. We found that abacavir and associated peptides bind to HLA-B*57:01 in a highly diverse range...
The conformational dynamism of proteins is well established. Rather than having a single structur... more The conformational dynamism of proteins is well established. Rather than having a single structure, proteins are more accurately described as a conformational ensemble that exists across a rugged energy landscape, where different conformational sub-states interconvert. The interaction between αβ T cell receptors (TCR) and cognate peptide-MHC (pMHC) is no exception, and is a dynamic process that involves substantial conformational change. This review focuses on technological advances that have begun to establish the role of conformational dynamics and dynamic allostery in TCR recognition of the pMHC and the early stages of signaling. We discuss how the marriage of molecular dynamics (MD) simulations with experimental techniques provides us with new ways to dissect and interpret the process of TCR ligation. Notably, application of simulation techniques lags behind other fields, but is predicted to make substantial contributions. Finally, we highlight integrated approaches that are being used to shed light on some of the key outstanding questions in the early events leading to TCR signaling.
Canonical mechanisms of protein evolution include the duplication and diversification of pre-exis... more Canonical mechanisms of protein evolution include the duplication and diversification of pre-existing folds through genetic alterations that include point mutations, insertions, deletions, and copy number amplifications, as well as post-translational modifications that modify processes such as folding efficiency and cellular localization. Following a survey of the human mutation database, we have identified an additional mechanism that we term "structural capacitance," which results in the de novo generation of microstructure in previously disordered regions. We suggest that the potential for structural capacitance confers select proteins with the capacity to evolve over rapid timescales, facilitating saltatory evolution as opposed to gradualistic canonical Darwinian mechanisms. Our results implicate the elements of protein microstructure generated by this distinct mechanism in the pathogenesis of a wide variety of human diseases. The benefits of rapidly furnishing the pot...
The Journal of experimental medicine, Jan 11, 2016
Viral infection activates danger signals that are transmitted via the retinoic acid-inducible gen... more Viral infection activates danger signals that are transmitted via the retinoic acid-inducible gene 1-like receptor (RLR), nucleotide-binding oligomerization domain-like receptor (NLR), and Toll-like receptor (TLR) protein signaling cascades. This places host cells in an antiviral posture by up-regulating antiviral cytokines including type-I interferon (IFN-I). Ubiquitin modifications and cross-talk between proteins within these signaling cascades potentiate IFN-I expression, and inversely, a growing number of viruses are found to weaponize the ubiquitin modification system to suppress IFN-I. Here we review how host- and virus-directed ubiquitin modification of proteins in the RLR, NLR, and TLR antiviral signaling cascades modulate IFN-I expression.
CD1d is a non-polymorphic antigen-presenting molecule that is structurally related to the MHC cla... more CD1d is a non-polymorphic antigen-presenting molecule that is structurally related to the MHC class I molecule. CD1d molecules are heterodimers that are composed of a heavy chain that is non-covalently associated with β2-microglobulin (β2m). The heavy chain is composed of three extracellular domains designated α1, α2 and α3 as well as a transmembrane and an intracytoplasmic domain. The antigen-binding cavity is formed from the α1 and α2 domains of the heavy chain. These domains comprise two α-helices which flank the opening of the cleft and a β-sheet floor (see Fig. 1a and b). and Jamie Rossjohn
The energetic bases of T cell recognition are unclear. Here, we studied the &amp;amp;amp;amp;... more The energetic bases of T cell recognition are unclear. Here, we studied the &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;energetic landscape&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; of peptide-major histocompatibility complex (pMHC) recognition by an immunodominant alphabeta T cell receptor (TCR). We quantified and evaluated the effect of natural and systematic substitutions in the complementarity-determining region (CDR) loops on ligand binding in the context of the structural detail of each component of the immunodominant TCR-pMHC complex. The CDR1 and CDR2 loops contributed minimal energy through direct recognition of the antigen and instead had a chief function in stabilizing the ligated CDR3 loops. The underlying energetic basis for recognition lay in the CDR3 loops. Therefore the energetic burden of the CDR loops in the TCR-pMHC interaction is variable among TCRs, reflecting the inherent adaptability of the TCR in ligating different ligands.
The innate immune response to viruses is critical for the correct establishment of protective ada... more The innate immune response to viruses is critical for the correct establishment of protective adaptive immunity. Amongst the many pathways involved, the NLRP3 [nucleotide-binding oligomerisation domain (NOD)-like receptor protein 3 (NLRP3)] inflammasome has received considerable attention, particularly in the context of immunity and pathogenesis during infection with influenza A (IAV) and SARS-CoV-2, the causative agent of COVID-19. Activation of the NLRP3 inflammasome results in the secretion of the proinflammatory cytokines IL-1β and IL-18, commonly coupled with pyroptotic cell death. While this mechanism is protective and key to host defense, aberrant NLRP3 inflammasome activation causes a hyperinflammatory response and excessive release of cytokines, both locally and systemically. Here, we discuss key molecules in the NLRP3 pathway that have also been shown to have significant roles in innate and adaptive immunity to viruses, including DEAD box helicase X-linked (DDX3X), vimenti...
RIG-I (retinoic-acid inducible gene I, also known as DDX58) is the best characterized receptor wi... more RIG-I (retinoic-acid inducible gene I, also known as DDX58) is the best characterized receptor within the RIG-I like receptor (RLR) family. Together with MDA5 (melanoma differentiation-associated 5) and LGP2 (laboratory of genetics and physiology 2), this family of cytoplasmic pattern recognition receptors (PRRs) are sentinels for intracellular viral RNA that is a product of viral infection. The RLR receptors provide frontline defence against viral infections in most tissues. RLR ligands The RIG-I receptor prefers to bind short (<2000 bp) single-or double-stranded RNA carrying an uncapped 5' triphosphate and additional motifs such as poly-uridine rich RNA motifs. [1][2] RIG-I triggers an immune response to RNA viruses from various families including the paramyxoviruses (e.g. measles), rhabdoviruses (e.g. vesicular stomatitis virus) and orthomyxoviruses (e.g. influenza A). [3][4][5][6][7] MDA5 ligands are poorly characterized, but the preference is for long double-stranded RNA (>2000 bp), such as the replicative form of picornavirus RNA that is found in picornavirus-infected cells. [8][9] LGP2 binds to blunt-ended double-stranded RNA of variable length, [10][11] and also to RNA-bound MDA5 to regulate filament formation. [12] The latter is linked to LGP2's recognition of picornaviruses (e.g. encephalomyocarditis virus), as per MDA5. [13]
Uncontrolled cell proliferation and genomic instability are common features of cancer and can ari... more Uncontrolled cell proliferation and genomic instability are common features of cancer and can arise from, respectively, the loss of cell-cycle control and defective checkpoints. Ubiquitin-mediated proteolysis, ultimately executed by ubiquitin-ligating enzymes (E3s), plays a key part in cell-cycle regulation and is dominated by two multisubunit E3s, the anaphase-promoting complex (or cyclosome) (APC/C) and SKP1–cullin-1–F-box (SCF) complex. We highlight the role of APC/C and the SCF bound to F-box proteins, FBXW7, SKP2, and β-TrCP, in regulating the abundance of select fundamental proteins, primarily during the cell cycle, that are associated with human cancer. The clinical success of the first proteasome inhibitor, bortezomib, in treating multiple myeloma and mantle-cell lymphoma set the precedent for viewing the ubiquitin–proteasome system as a druggable target for cancer. Given that there are more E3s than kinases, selective, small-molecule E3 inhibitors have the potential of open...
Dynamics plays an important but underappreciated role in the interaction between the T cell recep... more Dynamics plays an important but underappreciated role in the interaction between the T cell receptor (TCR) and peptide-bound major histocompatibility complex (pMHC). Crystallographic studies have provided key molecular insights into this interaction; however, due to inherent features of the structural approach, the image of TCR-pMHC interactions that has emerged is a static one. In this review, we discuss how molecular dynamics (MD) simulations can complement and extend current experimental methods aimed at examining TCR-pMHC dynamics. We review the insights obtained from studies that leverage MD approaches, and propose that an integrative strategy that harnesses both MD simulations and structural and biophysical methods will provide new inroads into understanding the transitory and dynamic molecular events that dictate TCR signaling and T cell activation.
Viral disease is one of the greatest burdens for human health worldwide, with an urgent need for ... more Viral disease is one of the greatest burdens for human health worldwide, with an urgent need for efficacious antiviral strategies. While antiviral drugs are available, in many cases, they are prone to the development of drug resistance. A way to overcome drug resistance associated with common antiviral therapies is to develop antivirals targeting host cellular co-factors critical to viral replication, such as DEAD-box helicase 3 X-linked (DDX3X), which plays key roles in RNA metabolism and the antiviral response. Here, we use biochemical/biophysical approaches and infectious assays to show for the first time that the small molecule RK-33 has broad-spectrum antiviral action by inhibiting the enzymatic activities of DDX3X. Importantly, we show that RK-33 is efficacious at low micromolar concentrations in limiting infection by human parainfluenza virus type 3 (hPIV-3), respiratory syncytial virus (RSV), dengue virus (DENV), Zika virus (ZIKV) or West Nile virus (WNV)—for all of which, n...
The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon ... more The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon (IFN) production and antiviral gene expression in response to RNA virus infection. Consequently, RLR signalling is tightly regulated by both host and viral factors. Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade, playing both ubiquitination-dependent and -independent roles in RIG-I-mediated IFN induction. However, additional regulatory roles are emerging. Here, we show a novel interaction between TRIM25 and another protein in the RLR pathway that is essential for type I IFN induction, DEAD-box helicase 3X (DDX3X). In vitro assays and knockdown studies reveal that TRIM25 ubiquitinates DDX3X at lysine 55 (K55) and that TRIM25 and DDX3X cooperatively enhance IFNB1 induction following RIG-I activation, but the latter is independent of TRIM25’s catalytic activity. Furthermore, we found that the influenza A virus no...
The transport of host proteins into and out of the nucleus is key to host function. However, nucl... more The transport of host proteins into and out of the nucleus is key to host function. However, nuclear transport is restricted by nuclear pores that perforate the nuclear envelope. Protein intrinsic disorder is an inherent feature of this selective transport barrier and is also a feature of the nuclear transport receptors that facilitate the active nuclear transport of cargo, and the nuclear transport signals on the cargo itself. Furthermore, intrinsic disorder is an inherent feature of viral proteins and viral strategies to disrupt host nucleocytoplasmic transport to benefit their replication. In this review, we highlight the role that intrinsic disorder plays in the nuclear transport of host and viral proteins. We also describe viral subversion mechanisms of the host nuclear transport machinery in which intrinsic disorder is a feature. Finally, we discuss nuclear import and export as therapeutic targets for viral infectious disease.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
DEAD-box helicase 3, X-linked (DDX3X) regulates the retinoic acid-inducible gene I (RIG-I)-like r... more DEAD-box helicase 3, X-linked (DDX3X) regulates the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-mediated antiviral response, but can also be a host factor contributing to the replication of viruses of significance to human health, such as human immunodeficiency virus type 1 (HIV-1). These roles are mediated in part through its ability to actively shuttle between the nucleus and the cytoplasm to modulate gene expression, although the trafficking mechanisms, and impact thereof on immune signaling and viral infection, are incompletely defined. We confirm that DDX3X nuclear export is mediated by the nuclear transporter exportin-1/CRM1, dependent on an N-terminal, leucine-rich nuclear export signal (NES) and the monomeric guanine nucleotide binding protein Ran in activated GTP-bound form. Transcriptome profiling and ELISA show that exportin-1-dependent export of DDX3X to the cytoplasm strongly impacts IFN-β production and the upregulation of immune genes in response to inf...
A structural characterization of the interaction between T cell receptors (TCR) and cognate pepti... more A structural characterization of the interaction between T cell receptors (TCR) and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the X-ray data. We found that in a large number of systems ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship be...
Dengue virus (DENV) threatens almost 70% of the world’s population, with no effective vaccine or ... more Dengue virus (DENV) threatens almost 70% of the world’s population, with no effective vaccine or therapeutic currently available. A key contributor to infection is nuclear localisation in the infected cell of DENV nonstructural protein 5 (NS5) through the action of the host importin (IMP) α/β1 proteins. Here, we used a range of microscopic, virological and biochemical/biophysical approaches to show for the first time that the small molecule GW5074 has anti-DENV action through its novel ability to inhibit NS5–IMPα/β1 interaction in vitro as well as NS5 nuclear localisation in infected cells. Strikingly, GW5074 not only inhibits IMPα binding to IMPβ1, but can dissociate preformed IMPα/β1 heterodimer, through targeting the IMPα armadillo (ARM) repeat domain to impact IMPα thermal stability and α-helicity, as shown using analytical ultracentrifugation, thermostability analysis and circular dichroism measurements. Importantly, GW5074 has strong antiviral activity at low µM concentrations...
A structural characterization of the interaction between ab TCRs and cognate peptide-MHC (pMHC) i... more A structural characterization of the interaction between ab TCRs and cognate peptide-MHC (pMHC) is central to understanding adaptive T cell-mediated immunity. X-ray crystallography, although the source of much structural data, traditionally provides only a static snapshot of the protein. Given the emerging evidence for the important role of conformational dynamics in protein function, we interrogated 309 crystallographic structures of pMHC complexes using ensemble refinement, a technique that can extract dynamic information from the x-ray data. Focusing on a subset of human pMHC class I systems, we found that in many cases, ensemble methods were able to uncover previously hidden evidence of significant conformational plasticity, thereby revealing additional information that can build upon and significantly enhance functional interpretations that are based on a single static structure. Notable examples include the interpretation of differences in the disease association of HLA subtypes, the relationship between peptide prominence and TCR recognition, the role of conformational flexibility in vaccine design, and the discrimination between induced fit and conformational selection models of TCR binding. We show that the currently widespread practice of analyzing pMHC interactions via the study of a single crystallographic structure does not make use of pertinent and easily accessible information from x-ray data concerning alternative protein conformations. This new analysis therefore not only highlights the capacity for ensemble methods to significantly enrich the interpretation of decades of structural data but also provides previously missing information concerning the dynamics of existing characterized TCR-pMHC interactions.
Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication ... more Abacavir is an antiretroviral drug used to reduce human immunodeficiency virus (HIV) replication and decrease the risk of developing acquired immune deficiency syndrome (AIDS). However, its therapeutic value is diminished by the fact that it is associated with drug hypersensitivity reactions in up to 8% of treated patients. This hypersensitivity is strongly associated with patients carrying human leukocyte antigen (HLA)-B*57:01, but not patients carrying closely related alleles. Abacavir’s specificity to HLA-B*57:01 is attributed to its binding site within the peptide-binding cleft and subsequent influence of the repertoire of peptides that can bind HLA-B*57:01. To further our understanding of abacavir-induced hypersensitivity we used molecular dynamics (MD) to analyze the dynamics of three different peptides bound to HLA-B*57:01 in the presence and absence of abacavir or abacavir analogues. We found that abacavir and associated peptides bind to HLA-B*57:01 in a highly diverse range...
The conformational dynamism of proteins is well established. Rather than having a single structur... more The conformational dynamism of proteins is well established. Rather than having a single structure, proteins are more accurately described as a conformational ensemble that exists across a rugged energy landscape, where different conformational sub-states interconvert. The interaction between αβ T cell receptors (TCR) and cognate peptide-MHC (pMHC) is no exception, and is a dynamic process that involves substantial conformational change. This review focuses on technological advances that have begun to establish the role of conformational dynamics and dynamic allostery in TCR recognition of the pMHC and the early stages of signaling. We discuss how the marriage of molecular dynamics (MD) simulations with experimental techniques provides us with new ways to dissect and interpret the process of TCR ligation. Notably, application of simulation techniques lags behind other fields, but is predicted to make substantial contributions. Finally, we highlight integrated approaches that are being used to shed light on some of the key outstanding questions in the early events leading to TCR signaling.
Canonical mechanisms of protein evolution include the duplication and diversification of pre-exis... more Canonical mechanisms of protein evolution include the duplication and diversification of pre-existing folds through genetic alterations that include point mutations, insertions, deletions, and copy number amplifications, as well as post-translational modifications that modify processes such as folding efficiency and cellular localization. Following a survey of the human mutation database, we have identified an additional mechanism that we term "structural capacitance," which results in the de novo generation of microstructure in previously disordered regions. We suggest that the potential for structural capacitance confers select proteins with the capacity to evolve over rapid timescales, facilitating saltatory evolution as opposed to gradualistic canonical Darwinian mechanisms. Our results implicate the elements of protein microstructure generated by this distinct mechanism in the pathogenesis of a wide variety of human diseases. The benefits of rapidly furnishing the pot...
The Journal of experimental medicine, Jan 11, 2016
Viral infection activates danger signals that are transmitted via the retinoic acid-inducible gen... more Viral infection activates danger signals that are transmitted via the retinoic acid-inducible gene 1-like receptor (RLR), nucleotide-binding oligomerization domain-like receptor (NLR), and Toll-like receptor (TLR) protein signaling cascades. This places host cells in an antiviral posture by up-regulating antiviral cytokines including type-I interferon (IFN-I). Ubiquitin modifications and cross-talk between proteins within these signaling cascades potentiate IFN-I expression, and inversely, a growing number of viruses are found to weaponize the ubiquitin modification system to suppress IFN-I. Here we review how host- and virus-directed ubiquitin modification of proteins in the RLR, NLR, and TLR antiviral signaling cascades modulate IFN-I expression.
CD1d is a non-polymorphic antigen-presenting molecule that is structurally related to the MHC cla... more CD1d is a non-polymorphic antigen-presenting molecule that is structurally related to the MHC class I molecule. CD1d molecules are heterodimers that are composed of a heavy chain that is non-covalently associated with β2-microglobulin (β2m). The heavy chain is composed of three extracellular domains designated α1, α2 and α3 as well as a transmembrane and an intracytoplasmic domain. The antigen-binding cavity is formed from the α1 and α2 domains of the heavy chain. These domains comprise two α-helices which flank the opening of the cleft and a β-sheet floor (see Fig. 1a and b). and Jamie Rossjohn
The energetic bases of T cell recognition are unclear. Here, we studied the &amp;amp;amp;amp;... more The energetic bases of T cell recognition are unclear. Here, we studied the &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;energetic landscape&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; of peptide-major histocompatibility complex (pMHC) recognition by an immunodominant alphabeta T cell receptor (TCR). We quantified and evaluated the effect of natural and systematic substitutions in the complementarity-determining region (CDR) loops on ligand binding in the context of the structural detail of each component of the immunodominant TCR-pMHC complex. The CDR1 and CDR2 loops contributed minimal energy through direct recognition of the antigen and instead had a chief function in stabilizing the ligated CDR3 loops. The underlying energetic basis for recognition lay in the CDR3 loops. Therefore the energetic burden of the CDR loops in the TCR-pMHC interaction is variable among TCRs, reflecting the inherent adaptability of the TCR in ligating different ligands.
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