Structural Insights into the DNA-Binding Mechanism of BCL11A: The Integral Role of ZnF6.

The transcription factor BCL11A is a critical regulator of the switch from fetal hemoglobin (HbF: α 2 γ 2 ) to adult hemoglobin (HbA: α 2 ß 2 ) during development. BCL11A binds at a cognate recognition site (TGACCA) in the γ-globin gene promoter and represses its expression. DNA-binding is mediated by a triple zinc finger domain, designated ZnF456. Here, we report comprehensive investigation of ZnF456, leveraging X-ray crystallography and NMR to determine the structures in both the presence and absence of DNA. We delve into the dynamics and mode of interaction with DNA. Moreover, we discovered that the last zinc finger of BCL11A (ZnF6) plays a special role in DNA binding and γ-globin gene repression. Our findings help account for some rare γ-globin gene promoter mutations that perturb BCL11A binding and lead to increased HbF in adults (hereditary persistence of fetal hemoglobin). Comprehending the DNA binding mechanism of BCL11A opens avenues for the strategic, structure-based design of novel therapeutics targeting sickle cell disease and ß-thalassemia.

Viruses inhibit TIR gcADPR signalling to overcome bacterial defence.

The Toll/interleukin-1 receptor (TIR) domain is a key component of immune receptors that identify pathogen invasion in bacteria, plants and animals1-3. In the bacterial antiphage system Thoeris, as well as in plants, recognition of infection stimulates TIR domains to produce an immune signalling molecule whose molecular structure remains elusive. This molecule binds and activates the Thoeris immune effector, which then executes the immune function1. We identified a large family of phage-encoded proteins, denoted here as Thoeris anti-defence 1 (Tad1), that inhibit Thoeris immunity. We found that Tad1 proteins are 'sponges' that bind and sequester the immune signalling molecule produced by TIR-domain proteins, thus decoupling phage sensing from immune effector activation and rendering Thoeris inactive. Tad1 can also efficiently sequester molecules derived from a plant TIR-domain protein, and a high-resolution crystal structure of Tad1 bound to a plant-derived molecule showed a unique chemical structure of 1 ''-2' glycocyclic ADPR (gcADPR). Our data furthermore suggest that Thoeris TIR proteins produce a closely related molecule, 1''-3' gcADPR, which activates ThsA an order of magnitude more efficiently than the plant-derived 1''-2' gcADPR. Our results define the chemical structure of a central immune signalling molecule and show a new mode of action by which pathogens can suppress host immunity.

Structural analysis of human CEACAM1 oligomerization.

The human (h) CEACAM1 GFCC' face serves as a binding site for homophilic and heterophilic interactions with various microbial and host ligands. hCEACAM1 has also been observed to form oligomers and micro-clusters on the cell surface which are thought to regulate hCEACAM1-mediated signaling. However, the structural basis for hCEACAM1 higher-order oligomerization is currently unknown. To understand this, we report a hCEACAM1 IgV oligomer crystal structure which shows how GFCC' face-mediated homodimerization enables highly flexible ABED face interactions to arise. Structural modeling and nuclear magnetic resonance (NMR) studies predict that such oligomerization is not impeded by the presence of carbohydrate side-chain modifications. In addition, using UV spectroscopy and NMR studies, we show that oligomerization is further facilitated by the presence of a conserved metal ion (Zn++ or Ni++) binding site on the G strand of the FG loop. Together these studies provide biophysical insights on how GFCC' and ABED face interactions together with metal ion binding may facilitate hCEACAM1 oligomerization beyond dimerization.

Structural basis of the dynamic human CEACAM1 monomer-dimer equilibrium.

Human (h) carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) function depends upon IgV-mediated homodimerization or heterodimerization with host ligands, including hCEACAM5, hTIM-3, PD-1, and a variety of microbial pathogens. However, there is little structural information available on how hCEACAM1 transitions between monomeric and dimeric states which in the latter case is critical for initiating hCEACAM1 activities. We therefore mutated residues within the hCEACAM1 IgV GFCC' face including V39, I91, N97, and E99 and examined hCEACAM1 IgV monomer-homodimer exchange using differential scanning fluorimetry, multi-angle light scattering, X-ray crystallography and/or nuclear magnetic resonance. From these studies, we describe hCEACAM1 homodimeric, monomeric and transition states at atomic resolution and its conformational behavior in solution through NMR assignment of the wildtype (WT) hCEACAM1 IgV dimer and N97A mutant monomer. These studies reveal the flexibility of the GFCC' face and its important role in governing the formation of hCEACAM1 dimers and selective heterodimers.

Cryo-EM structure of an activated GPCR-G protein complex in lipid nanodiscs.

G-protein-coupled receptors (GPCRs) are the largest superfamily of transmembrane proteins and the targets of over 30% of currently marketed pharmaceuticals. Although several structures have been solved for GPCR-G protein complexes, few are in a lipid membrane environment. Here, we report cryo-EM structures of complexes of neurotensin, neurotensin receptor 1 and Gαi1ß1γ1 in two conformational states, resolved to resolutions of 4.1 and 4.2 Å. The structures, determined in a lipid bilayer without any stabilizing antibodies or nanobodies, reveal an extended network of protein-protein interactions at the GPCR-G protein interface as compared to structures obtained in detergent micelles. The findings show that the lipid membrane modulates the structure and dynamics of complex formation and provide a molecular explanation for the stronger interaction between GPCRs and G proteins in lipid bilayers. We propose an allosteric mechanism for GDP release, providing new insights into the activation of G proteins for downstream signaling.

Topological analysis of the gp41 MPER on lipid bilayers relevant to the metastable HIV-1 envelope prefusion state.

The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein (gp) 41 is an attractive vaccine target for elicitation of broadly neutralizing antibodies (bNAbs) by vaccination. However, current details regarding the quaternary structural organization of the MPER within the native prefusion trimer [(gp120/41)3] are elusive and even contradictory, hindering rational MPER immunogen design. To better understand the structural topology of the MPER on the lipid bilayer, the adjacent transmembrane domain (TMD) was appended (MPER-TMD) and studied. Membrane insertion of the MPER-TMD was sensitive both to the TMD sequence and cytoplasmic residues. Antigen binding of MPER-specific bNAbs, in particular 10E8 and DH511.2_K3, was significantly impacted by the presence of the TMD. Furthermore, MPER-TMD assembly into 10-nm diameter nanodiscs revealed a heterogeneous membrane array comprised largely of monomers and dimers, as enumerated by bNAb Fab binding using single-particle electron microscopy analysis, arguing against preferential trimeric association of native MPER and TMD protein segments. Moreover, introduction of isoleucine mutations in the C-terminal heptad repeat to induce an extended MPER α-helical bundle structure yielded an antigenicity profile of cell surface-arrayed Env variants inconsistent with that found in the native prefusion state. In line with these observations, electron paramagnetic resonance analysis suggested that 10E8 inhibits viral membrane fusion by lifting the MPER N-terminal region out of the viral membrane, mandating the exposure of residues that would be occluded by MPER trimerization. Collectively, our data suggest that the MPER is not a stable trimer, but rather a dynamic segment adapted for structural changes accompanying fusion.

A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity.

A substantial fraction of eukaryotic proteins is folded and modified in the endoplasmic reticulum (ER) prior to export and secretion. Proteins that enter the ER but fail to fold correctly must be degraded, mostly in a process termed ER-associated degradation (ERAD). Both protein folding in the ER and ERAD are essential for proper immune function. Several E2 and E3 enzymes localize to the ER and are essential for various aspects of ERAD, but their functions and regulation are incompletely understood. Here we identify and characterize single domain antibody fragments derived from the variable domain of alpaca heavy chain-only antibodies (VHHs or nanobodies) that bind to the ER-localized E2 UBC6e, an enzyme implicated in ERAD. One such VHH, VHH05 recognizes a 14 residue stretch and enhances the rate of E1-catalyzed ubiquitin E2 loading in vitroand interferes with phosphorylation of UBC6e in response to cell stress. Identification of the peptide epitope recognized by VHH05 places it outside the E2 catalytic core, close to the position of activation-induced phosphorylation on Ser184. Our data thus suggests a site involved in allosteric regulation of UBC6e's activity. This VHH should be useful not only to dissect the participation of UBC6e in ERAD and in response to cell stress, but also as a high affinity epitope tag-specific reagent of more general utility.

Conformational Stabilization and Rapid Labeling of a 29-Residue Peptide by a Small Molecule Reaction Partner.

A 29-residue peptide (MP01), identified by in vitro selection for reactivity with a small molecule perfluoroaromatic, was modified and characterized using experimental and computational techniques, with the goal of understanding the molecular basis of its reactivity. These studies identified a six-amino acid point mutant (MP01-Gen4) that exhibited a reaction rate constant of 25.8 ± 1.8 M-1 s-1 at pH 7.4 and room temperature, approximately 2 orders of magnitude greater than that of its progenitor sequence and 3 orders of magnitude greater than background cysteine reactivity. MP01-Gen4 appeared to be conformationally dynamic and exhibited several properties reminiscent of larger protein molecules, including denaturant-sensitive structure and reactivity. We believe the majority of the reaction rate enhancement can be attributed to interaction of MP01-Gen4 with the perfluoroaromatic probe, which was found to stabilize a helical conformation of both MP01-Gen4 and nonreactive Cys-to-Ser or Cys-to-Ala variants. These findings demonstrate the ability of dynamic peptides to access proteinlike reaction mechanisms and the potential of perfluoroaromatic functionality to stabilize small peptide folds.

High resolution X-ray and NMR structural study of human T-cell immunoglobulin and mucin domain containing protein-3.

T-cell immunoglobulin and mucin domain containing protein-3 (TIM-3) is an important immune regulator. Here, we describe a novel high resolution (1.7 Å) crystal structure of the human (h)TIM-3 N-terminal variable immunoglobulin (IgV) domain with bound calcium (Ca++) that was confirmed by nuclear magnetic resonance (NMR) spectroscopy. Significant conformational differences were observed in the B-C, C'-C″ and C'-D loops of hTIM-3 compared to mouse (m)TIM-3, hTIM-1 and hTIM-4. Further, the conformation of the C-C' loop of hTIM-3 was notably different from hTIM-4. Consistent with the known metal ion-dependent binding of phosphatidylserine (PtdSer) to mTIM-3 and mTIM-4, the NMR spectral analysis and crystal structure of Ca++-bound hTIM-3 revealed that residues in the hTIM-3 F-G loop coordinate binding to Ca++. In addition, we established a novel biochemical assay to define hTIM-3 functionality as determined by binding to human carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1). These studies provide new insights useful for understanding and targeting hTIM-3.

Covalently circularized nanodiscs for studying membrane proteins and viral entry.

We engineered covalently circularized nanodiscs (cNDs) which, compared with standard nanodiscs, exhibit enhanced stability, defined diameter sizes and tunable shapes. Reconstitution into cNDs enhanced the quality of nuclear magnetic resonance spectra for both VDAC-1, a ß-barrel membrane protein, and the G-protein-coupled receptor NTR1, an α-helical membrane protein. In addition, we used cNDs to visualize how simple, nonenveloped viruses translocate their genomes across membranes to initiate infection.

Solution Structure of the Cuz1 AN1 Zinc Finger Domain: An Exposed LDFLP Motif Defines a Subfamily of AN1 Proteins.

Zinc binding domains are common and versatile protein structural motifs that mediate diverse cellular functions. Among the many structurally distinct families of zinc finger (ZnF) proteins, the AN1 domain remains poorly characterized. Cuz1 is one of two AN1 ZnF proteins in the yeast S. cerevisiae, and is a stress-inducible protein that functions in protein degradation through direct interaction with the proteasome and Cdc48. Here we report the solution structure of the Cuz1 AN1 ZnF which reveals a compact C6H2 zinc-coordinating domain that resembles a two-finger hand holding a tri-helical clamp. A central phenylalanine residue sits between the two zinc-coordinating centers. The position of this phenylalanine, just before the penultimate zinc-chelating cysteine, is strongly conserved from yeast to man. This phenylalanine shows an exceptionally slow ring-flipping rate which likely contributes to the high rigidity and stability of the AN1 domain. In addition to the zinc-chelating residues, sequence analysis of Cuz1 indicates a second highly evolutionarily conserved motif. This LDFLP motif is shared with three human proteins-Zfand1, AIRAP, and AIRAP-L-the latter two of which share similar cellular functions with Cuz1. The LDFLP motif, while embedded within the zinc finger domain, is surface exposed, largely uninvolved in zinc chelation, and not required for the overall fold of the domain. The LDFLP motif was dispensable for Cuz1's major known functions, proteasome- and Cdc48-binding. These results provide the first structural characterization of the AN1 zinc finger domain, and suggest that the LDFLP motif may define a sub-family of evolutionarily conserved AN1 zinc finger proteins.

Generation of Long-Lived Bone Marrow Plasma Cells Secreting Antibodies Specific for the HIV-1 gp41 Membrane-Proximal External Region in the Absence of Polyreactivity.

UNLABELLED: An effective preventive vaccine is highly sought after in order to stem the current HIV-1 pandemic. Both conservation of contiguous gp41 membrane-proximal external region (MPER) amino acid sequences across HIV-1 clades and the ability of anti-MPER broadly neutralizing antibodies (BNAbs) to block viral hemifusion/fusion establish the MPER as a prime vaccination target. In earlier studies, we described the development of an MPER vaccine formulation that takes advantage of liposomes to array the MPER on a lipid bilayer surface, paralleling its native configuration on the virus membrane while also incorporating molecular adjuvant and CD4 T cell epitope cargo. Here we demonstrate that several immunizations with MPER/liposomes induce high levels of bone marrow long-lived plasma cell (LLPC) antibody production. Single-cell immunoglobulin gene retrieval analysis shows that these plasma cells are derived from a germ line repertoire of B cells with a diverse representation of immunoglobulin genes, exhibiting antigen-driven positive selection. Characterization of LLPC recombinant monoclonal antibodies (rMAbs) indicates that antigen recognition is achieved through convergence on a common epitopic focus by utilizing various complementarity-determining region H3 (CDRH3) lengths. Importantly, the vast majority of rMAbs produced from these cells lack polyreactivity yet manifest antigen specificity in the context of lipids, shaping MPER-specific paratopes through selective pressure. Taken together, these findings demonstrate that the MPER is a vaccine target with minimal risk of generating off-target autoimmunity. IMPORTANCE: A useful vaccine must generate desired long-term, antigen-specific antibody responses devoid of polyreactivity or autoreactivity. The common polyreactive features of some HIV-1 BNAbs have raised concern about elicitation of anti-MPER antibodies. Utilizing single-LLPC repertoire analysis and biophysical characterization of anti-MPER rMAbs, we show that their fine specificities require a structural fitness of the antibody combining site involving heavy and light chain variable domains shaped by somatic hypermutation and affinity maturation of B cells in the germinal center. Perhaps more importantly, our results demonstrate that the majority of MPER-specific antibodies are not inherently polyspecific and/or autoreactive, suggesting that polyreactivity of MPER-specific antibodies is separable from their antigen specificity.

NMR resonance assignments of the catalytic domain of human serine/threonine phosphatase calcineurin in unligated and PVIVIT-peptide-bound states.

Calcineurin (Cn) is a serine/threonine phosphatase that plays pivotal roles in many physiological processes. In T cell, Cn targets the nuclear factors of activated T-cell (NFATs), transcription factors that activate cytokine genes. Elevated intracellular calclium concentration activates Cn to dephosphorylate multiple serine residues within the NFAT regulatory domain, which triggers joint nuclear translocation of NFAT and Cn. This relies on the interaction between the catalytic domain of Cn (CnA) and the conserved PxIxIT motif. Here, we present the assignment of CnA resonances in unligated form and in complex with a 14-residue peptide containing a PVIVIT sequence that was derived from affinity driven peptide selection based on the conserved PxIxIT motif of NFATs. Although a complete assignment was not possible mainly due to the paramagnetic line broadening induced by an iron in the CnA catalytic center, the assignment was extensively verified by amino-acid selective labeling of Arg, Leu, Lys, and Val, which cover one third of the CnA residues. Nevertheless, the assignments were used to determine the structure of the CnA-PVIVIT peptide complex and provide the basis for investigation of the interactions of CnA with physiological interaction partners and small organic compounds that disrupt the Cn-NFAT interaction.

Disruption of helix-capping residues 671 and 674 reveals a role in HIV-1 entry for a specialized hinge segment of the membrane proximal external region of gp41.

HIV-1 (human immunodeficiency virus type 1) uses its trimeric gp160 envelope (Env) protein consisting of non-covalently associated gp120 and gp41 subunits to mediate entry into human T lymphocytes. A facile virus fusion mechanism compensates for the sparse Env copy number observed on viral particles and includes a 22-amino-acid, lentivirus-specific adaptation at the gp41 base (amino acid residues 662-683), termed the membrane proximal external region (MPER). We show by NMR and EPR that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via interconverting structures while, at the same time, affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell-cell fusion and hemifusion, as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix capping, thereby disrupting hinge function.

Immunogenicity of membrane-bound HIV-1 gp41 membrane-proximal external region (MPER) segments is dominated by residue accessibility and modulated by stereochemistry.

Structural characterization of epitope-paratope pairs has contributed to the understanding of antigenicity. By contrast, few structural studies relate to immunogenicity, the process of antigen-induced immune responses in vivo. Using a lipid-arrayed membrane-proximal external region (MPER) of HIV-1 glycoprotein 41 as a model antigen, we investigated the influence of physicochemical properties on immunogenicity in relation to structural modifications of MPER/liposome vaccines. Anchoring the MPER to the membrane via an alkyl tail or transmembrane domain retained the MPER on liposomes in vivo, while preserving MPER secondary structure. However, structural modifications that affected MPER membrane orientation and antigenic residue accessibility strongly impacted induced antibody responses. The solvent-exposed MPER tryptophan residue (Trp-680) was immunodominant, focusing immune responses, despite sequence variability elsewhere. Nonetheless, immunogenicity could be readily manipulated using site-directed mutagenesis or structural constraints to modulate amino acid surface display. These studies provide fundamental insights for immunogen design aimed at targeting B cell antibody responses.

TCR Mechanobiology: Torques and Tunable Structures Linked to Early T Cell Signaling.

Mechanotransduction is a basis for receptor signaling in many biological systems. Recent data based upon optical tweezer experiments suggest that the TCR is an anisotropic mechanosensor, converting mechanical energy into biochemical signals upon specific peptide-MHC complex (pMHC) ligation. Tangential force applied along the pseudo-twofold symmetry axis of the TCR complex post-ligation results in the αß heterodimer exerting torque on the CD3 heterodimers as a consequence of molecular movement at the T cell-APC interface. Accompanying TCR quaternary change likely fosters signaling via the lipid bilayer predicated on the magnitude and direction of the TCR-pMHC force. TCR glycans may modulate quaternary change, thereby altering signaling outcome as might the redox state of the CxxC motifs located proximal to the TM segments in the heterodimeric CD3 subunits. Predicted alterations in TCR TM segments and surrounding lipid will convert ectodomain ligation into the earliest intracellular signaling events.

Antibody mechanics on a membrane-bound HIV segment essential for GP41-targeted viral neutralization.

Broadly neutralizing antibodies such as 2F5 are directed against the membrane-proximal external region (MPER) of HIV-1 GP41 and recognize well-defined linear core sequences. These epitopes can be engrafted onto protein scaffolds to serve as immunogens with high structural fidelity. Although antibodies that bind to this core GP41 epitope can be elicited, they lack neutralizing activity. To understand this paradox, we used biophysical methods to investigate the binding of human 2F5 to the MPER in a membrane environment, where it resides in vivo. Recognition is stepwise, through a paratope more extensive than core binding site contacts alone, and dynamic rearrangement through an apparent scoop-like movement of heavy chain complementarity-determining region 3 (CDRH3) is essential for MPER extraction from the viral membrane. Core-epitope recognition on the virus requires the induction of conformational changes in both the MPER and the paratope. Hence, target neutralization through this lipid-embedded viral segment places stringent requirements on the plasticity of the antibody combining site.

Structure of the VP16 transactivator target in the Mediator.

The human Mediator coactivator complex interacts with many transcriptional activators and facilitates recruitment of RNA polymerase II to promote target gene transcription. The MED25 subunit is a critical target of the potent herpes simplex 1 viral transcriptional activator VP16. Here we determine the solution structure of the MED25 VP16-binding domain (VBD) and define its binding site for the N-terminal portion of the VP16 transactivation domain (TADn). A hydrophobic furrow, formed by a ß-barrel and two α-helices in MED25 VBD, interacts tightly with VP16 TADn. Mutations in this furrow prevent binding of VP16 TAD to MED25 VBD and interfere with the ability of overexpressed MED25 VBD to inhibit VP16-dependent transcriptional activation in vivo. This detailed molecular understanding of transactivation by the benchmark activator VP16 could provide important insights into viral and cellular gene activation mechanisms.

Distinctive CD3 heterodimeric ectodomain topologies maximize antigen-triggered activation of alpha beta T cell receptors.

The alphabeta TCR has recently been suggested to function as an anisotropic mechanosensor during immune surveillance, converting mechanical energy into a biochemical signal upon specific peptide/MHC ligation of the alphabeta clonotype. The heterodimeric CD3epsilongamma and CD3epsilondelta subunits, each composed of two Ig-like ectodomains, form unique side-to-side hydrophobic interfaces involving their paired G-strands, rigid connectors to their respective transmembrane segments. Those dimers are laterally disposed relative to the alphabeta heterodimer within the TCR complex. In this paper, using structure-guided mutational analysis, we investigate the functional consequences of a striking asymmetry in CD3gamma and CD3delta G-strand geometries impacting ectodomain shape. The uniquely kinked conformation of the CD3gamma G-strand is crucial for maximizing Ag-triggered TCR activation and surface TCR assembly/expression, offering a geometry to accommodate juxtaposition of CD3gamma and TCR beta ectodomains and foster quaternary change that cannot be replaced by the isologous CD3delta subunit's extracellular region. TCRbeta and CD3 subunit protein sequence analyses among Gnathostomata species show that the Cbeta FG loop and CD3gamma subunit coevolved, consistent with this notion. Furthermore, restoration of T cell activation and development in CD3gamma(-/-) mouse T lineage cells by interspecies replacement can be rationalized from structural insights on the topology of chimeric mouse/human CD3epsilondelta dimers. Most importantly, our findings imply that CD3gamma and CD3delta evolved from a common precursor gene to optimize peptide/MHC-triggered alphabeta TCR activation.

Autoinhibitory interaction in the multidomain adaptor protein Nck: possible roles in improving specificity and functional diversity.

Nck is a functionally versatile multidomain adaptor protein consisting of one SH2 and three SH3 domains. In most cases, the SH2 domain mediates binding to tyrosine-phosphorylated receptors or cytosolic proteins, which leads to the formation of larger protein complexes via the SH3 domains. Nck plays a pivotal role in T-cell receptor-mediated reorganization of the actin cytoskeleton as well as in the formation of the immunological synapses. The modular domain structure and the functionality of the individual domains suggest that they might act independently. Here we report an interesting intramolecular interaction within Nck that occurs between a noncanonical yet conserved (K/R)x(K/R)RxxS sequence in the linker between the first and second SH3 domain (SH3.1/SH3.2) and the second SH3 domain (SH3.2). Because this interaction masks the proline-rich sequence binding site of the SH3.2 domain, the intramolecular interaction is self-inhibitory. This intramolecular interaction could, at least partially, explain the remarkable specificity of Nck toward proteins with proline-rich sequences. It may prevent nonspecific low-affinity binding while keeping the site available for high-affinity bivalent ligands that can bind multiple sites in Nck. This indicates that Nck does not simply adopt a "beads on a string" architecture but incorporates a higher-order organization for improved specificity and functionality.