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: αγ) to adult hemoglobin (HbA: αβ) 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.

Protein NMR assignment by isotope pattern recognition.

The current standard method for amino acid signal identification in protein NMR spectra is sequential assignment using triple-resonance experiments. Good software and elaborate heuristics exist, but the process remains laboriously manual. Machine learning does help, but its training databases need millions of samples that cover all relevant physics and every kind of instrumental artifact.

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 γ ) to adult hemoglobin (HbA: α β ) 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.

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.

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 animals. 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 function.

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.

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 Gαβγ in two conformational states, resolved to resolutions of 4.

Direct Tumor Killing and Immunotherapy through Anti-SerpinB9 Therapy.

Cancer therapies kill tumors either directly or indirectly by evoking immune responses and have been combined with varying levels of success. Here, we describe a paradigm to control cancer growth that is based on both direct tumor killing and the triggering of protective immunity. Genetic ablation of serine protease inhibitor SerpinB9 (Sb9) results in the death of tumor cells in a granzyme B (GrB)-dependent manner.

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.

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.

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.

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 β-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.

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.

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.

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.

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 αβ heterodimer exerting torque on the CD3 heterodimers as a consequence of molecular movement at the T cell-APC interface.

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.

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).

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.

Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments.

Heteronuclear direct-detection experiments, which utilize the slower relaxation properties of low gamma nuclei, such as (13)C have recently been proposed for sequence-specific assignment and structural analyses of large, unstructured, and/or paramagnetic proteins. Here we present two novel (15)N direct-detection experiments. The CAN experiment sequentially connects amide (15)N resonances using (13)C(alpha) chemical shift matching, and the CON experiment connects the preceding (13)C' nuclei.

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.