Human glycoprotein hormones such as thyroid-stimulating hormone (TSH) and follicle-stimulating hormone (FSH) belong to a broader family of cystine-knot hormones (CKHs), all of which act through leucine-rich-repeat (LRR)-containing G protein-coupled receptors (LGRs) with which they have coevolved from evolutionary predecessors in metazoan animals. There is substantial evidence for LGR dimers being required in the transmission of G-protein signals elicited by mammalian CKHs acting on their cognate LGRs. Yet, human LGRs are monomeric as extracted from cell membranes and also in cryo-EM structures, both when in apo, inactive state and when hormone bound in an LRR-elevated active state. Fortunately, the LGR from the nematode Caenorhabditis elegans (CeLGR) remains dimeric as detergent extracted for structure determination. In this study, we synthesize structural information from CeLGR, HsTSHR, and the other human LGRs together with biophysical evidence about physiological dimers to produce a theoretical description of conformational equilibria involved in CKH activation of LGRs. We develop a theory for the equilibria among conformations that govern signal transmission from hormone to G protein, we define the transitions of receptor activation in quantifiable terms, and we build and validate energetically feasible models for CeLGR and HsTSHR in their relevant 0:2, 1:2, and 2:2 hormone:receptor complexes. These developments provide a framework for understanding of signaling through CKH receptors and for devising structure-based hypotheses to test such conceptions.

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. When administrated along with the anti-CoRBS 17b, CJF-III-288 delayed viral rebound after ART interruption in HIV-1-infected humanized mice, demonstrating potential for eliciting ADCC in vivo. Structural and conformational analyses reveal that CJF-III-288, in combination with this anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation. This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

The neuroendocrine system that comprises the glycoprotein hormones (GpHs) and their receptors is essential for reproduction and metabolism. Each GpH hormone is an αβ heterodimer of cystine-knot proteins and its cognate receptor is a G-protein coupled receptor (GPCR) distinguished by a large leucine-rich-repeat (LRR) extracellular domain that binds the hormone and a class A GPCR transmembrane domain that signals through an associating heterotrimeric G protein. Hence, the receptors are called LRR-containing GPCRs-LGRs. The vertebrate GpHs and LGRs have co-evolved from homologs in the earliest metazoan animals, including sponges and comb jellies, but these are absent from unicellular organisms and plants. The two GpH subunits and accompanying LGR receptor of the nematode Caenorhabditis elegans are representative of the invertebrate evolutionary predecessors of human GpH proteins and their receptors, for example follicle-stimulating hormone (FSH) and the FSH receptor (FSHR). Atomic structures of the human GpHs and their receptors, which have been determined by X-ray crystallography and cryogenic electron microscopy (cryo-EM), inform the evolutionary process and provide a mechanistic understanding of the transmission of biochemical signals of hormone binding at the cell surface to the elicitation of second messengers such as cyclic AMP in the cytoplasm. There is compelling biochemical and cellular evidence for the importance of receptor dimers in GpH signaling in cells; yet, all of the human receptors are monomeric as defined beautifully by cryo-EM. Fortunately, the LGR of C. elegans is a stable dimer and its structure, when analyzed in the context of structural information from the human counterparts, predicts a hypothetical model for functionally relevant dimeric associations of the human GpH receptors.

The nuclear receptor PPARγ is a primary therapeutic target for insulin resistance and type 2 diabetes (T2D); however, its thiazolidinedione (TZD) class of PPARγ agonists have substantial safety concerns in clinical utilization. Genetic inhibition of PPARγ acetylation at K268 and K293 dissociates the major adverse effects of TZDs from insulin sensitization and other metabolic improvements. We therefore posit that chemical inhibition of PPARγ acetylation would elicit insulin sensitization with improved safety. Here we describe the identification of a synthetic thiopyrimidine derivative (TPMD) that acts as a small molecule inhibitor of PPARγ acetylation. TPMD improves insulin sensitivity, promotes brown remodeling of white adipose tissue (WAT), increases energy expenditure, and decreases adiposity in dietary and genetic mouse models of T2D. Importantly, TPMD is deprived of the major side effects of TZDs, including weight gain, fluid retention, cardiac hypertrophy, bone marrow adiposity, and bone loss. X-ray crystallography at 2.0 Å resolution reveals a unique binding mode of TPMD molecules to the PPARγ ligand-binding domain and provides structural and molecular basis for the acetylation inhibitory activity of TPMD together with site-directed mutagenesis studies. These findings identify TPMD as a first-in-class compound that specifically targets PPARγ acetylation and possesses the potential of developing into a safe insulin sensitizer to treat and prevent T2D and obesity.

The amino acid sequence of the tryptophan-rich sensory proteins (TSPO) is substantially conserved throughout all kingdoms of life. Human mitochondrial TSPO1 (HsTSPO1) binds to porphyrins and steroids, although its interactions with these molecules remains unknown. HsTSPO1 is associated with numerous physiological and pathological disorders, but the underlying molecular mechanisms are unknown. Here, we disclose the finding of human mitochondrial TSPO as a cholesterol-dependent protoporphyrin IX oxygenase. The results of our biochemical characterization are consistent with structural data and evolutionary analysis. The dependence of HsTSPO1 activity on cholesterol may be the result of the coevolution of this membrane protein with the membrane system. Our study provides a molecular foundation for comprehending the various roles played by mitochondrial TSPO in normal physiological and pathological situations.

Molecular replacement (MR) is highly effective for biomolecular crystal structure determination, increasingly so as the database of known structures has increased. For candidates without recognizable similarity to known structures, however, crystal structure analyses have nearly always required experiments for de novo phase evaluation. Now, with the unprecedented accuracy of AlphaFold predictions of protein structures from amino-acid sequences, an appreciable expansion of the reach of MR for proteins is realized. Here, we sought to automate an AlphaFold-guided MR procedure that tailors predictions to the MR problem at hand. We first optimized the reliability cutoff parameters for residue inclusion as tested in application to a previously MR-intractable problem. We then examined cases where AlphaFold by default predicts a conformation alternative to that of the candidate structure, devising tests for MR solution either from domain-specific predictions or from predictions based on diverse sequence subclusters. We tested subclustering procedures on an enzyme system that entails multiple MR-challenging conformations. The overall process as implemented in Phenix automatically surveys a succession of trials of increasing computational complexity until an MR solution is found or the options are exhausted. Validated MR solutions were found for 92% of one set of 158 challenging problems from the PDB and 93% of those from a second set of 215 challenges. Thus, many crystal structure analyses that previously required experimental phase evaluation can now be solved by AlphaFold-guided MR. In effect, this and related MR approaches are de novo phasing methods.

Glycoprotein hormones (GpHs) produced in the human pituitary act through receptors (GpHRs) in the gonads to support reproduction and in the thyroid for metabolism. GpHs are heterodimeric cystine-knot proteins; their receptors bind cognate hormones at an extracellular domain and signal through a transmembrane domain to heterotrimeric G proteins. GpHs and GpHRs have co-evolved from invertebrate counterparts. Structures of the human receptors as isolated for cryogenic electron microscopy (cryo-EM) are all monomeric despite compelling evidence for their functioning as dimers. Here we characterize the homologous receptor from Caenorhabditis elegans. Its biochemical properties are notably similar to those of the thyroid stimulating hormone receptor (TSHR) of humans. Structurally, it is an asymmetric dimer (protomers screw-transformed by 142°/4.1 Å), composed such that only one hormone could bind. This is compatible with the 1:2 asymmetry of negatively cooperative TSH:TSHR complexes and for the transactivation evident from functional complementation of binding-deficient and signaling-deficient GpHRs. By modeling, a symmetrized dimer can bind two hormones as in the 2:2 complexes that support TSHR switches in G-protein usage.

Stomata in leaves regulate gas (carbon dioxide and water vapor) exchange and water transpiration between plants and the atmosphere. SLow Anion Channel 1 (SLAC1) mediates anion efflux from guard cells and plays a crucial role in controlling stomatal aperture. It serves as a central hub for multiple signaling pathways in response to environmental stimuli, with its activity regulated through phosphorylation via various plant protein kinases. However, the molecular mechanism underlying SLAC1 phosphoactivation has remained elusive. Through a combination of protein sequence analyses, AlphaFold-based modeling and electrophysiological studies, we unveiled that the highly conserved motifs on the N- and C-terminal segments of SLAC1 form a cytosolic regulatory domain (CRD) that interacts with the transmembrane domain(TMD), thereby maintaining the channel in an autoinhibited state. Mutations in these conserved motifs destabilize the CRD, releasing autoinhibition in SLAC1 and enabling its transition into an activated state. Our further studies demonstrated that SLAC1 activation undergoes an autoinhibition-release process and subsequent structural changes in the pore helices. These findings provide mechanistic insights into the activation mechanism of SLAC1 and shed light on understanding how SLAC1 controls stomatal closure in response to environmental stimuli.

Malignant hyperthermia (MH) is a life-threatening pharmacogenetic condition triggered by volatile anesthetics, which activate pathogenic RyR1 mutants. The small molecule therapeutic dantrolene has long been used to treat MH. However, the binding site and mechanism of dantrolene remain unclear. Here, we present cryo-EM structures of RyR1 bound to dantrolene and the MH trigger agent 4-chloro-m-cresol (4CmC), revealing the dantrolene and 4CmC binding sites in atomic detail. Dantrolene binds stacked with ATP or ADP in the RY12 domain at the corner of the receptor, inducing a conformational change in this domain which is allosterically coupled to pore closure. Functional analyses revealed that ATP or ADP was required for dantrolene inhibition, and a single point mutation that disrupts the peripheral ATP binding site abolished ATP/ADP-dependent dantrolene inhibition. Strikingly, in the absence of dantrolene, this site selectively binds two ADP molecules, suggesting a possible role in ATP/ADP ratio sensing.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each G alpha subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of G alpha. We identified a single G alpha residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three G alpha subtypes, thereby conferring capacity for promiscuous G-protein coupling. Structures of the human calcium-sensing receptor can be bound into complex with G proteins from three different G alpha subtypes while maintaining G-protein-binding specificity.

HIV-1 envelope glycoproteins (Env) from primary HIV-1 isolates typically adopt a pretriggered "closed" conformation that resists to CD4-induced (CD4i) non-neutralizing antibodies (nnAbs) mediating antibody-dependent cellular cytotoxicity (ADCC). CD4-mimetic compounds (CD4mcs) "open-up" Env allowing binding of CD4i nnAbs, thereby sensitizing HIV-1-infected cells to ADCC. Two families of CD4i nnAbs, the anti-cluster A and anti-coreceptor binding site (CoRBS) Abs, are required to mediate ADCC in combination with the indane CD4mc BNM-III-170. Recently, new indoline CD4mcs with improved potency and breadth have been described. Here, we show that the lead indoline CD4mc, CJF-III-288, sensitizes HIV-1-infected cells to ADCC mediated by anti-CoRBS Abs alone, contributing to improved ADCC activity. Structural and conformational analyses reveal that CJF-III-288, in combination with anti-CoRBS Abs, potently stabilizes an asymmetric "open" State-3 Env conformation, This Env conformation orients the anti-CoRBS Ab to improve ADCC activity and therapeutic potential.

The marvel of X-ray crystallography is the beauty and precision of the atomic structures deduced from diffraction patterns. Since these patterns record only amplitudes, phases for the diffracted waves must also be evaluated for systematic structure determination. Thus, we have the phase problem as a central complication, both intellectually for the field and practically so for many analyses. Here, I discuss how we – myself, my laboratory and the diffraction community – have faced the phase problem, considering the evolution of methods for phase evaluation as structural biology developed to the present day. During the explosive growth of macromolecular crystallography, practice in diffraction analysis evolved from a universal reliance on isomorphous replacement to the eventual domination of anomalous diffraction for de novo structure determination. As the Protein Data Bank (PDB) grew and familial relationships among proteins became clear, molecular replacement overtook all other phasing methods; however, experimental phasing remained essential for molecules without obvious precedents, with multi- and single-wavelength anomalous diffraction (MAD and SAD) predominating. While the mathematics-based direct methods had proved to be inadequate for typical macromolecules, they returned to crack substantial selenium substructures in SAD analyses of selenomethionyl proteins. Native SAD, exploiting the intrinsic S and P atoms of biomolecules, has become routine. Selenomethionyl SAD and MAD were the mainstays of structural genomics efforts to populate the PDB with novel proteins. A recent dividend has been paid in the success of PDB-trained artificial intelligence approaches for protein structure prediction. Currently, molecular replacement with AlphaFold models often obviates the need for experimental phase evaluation. For multiple reasons, we are now unfazed by the phase problem. Cryo-EM analysis is an attractive alternative to crystallography for many applications faced by today's structural biologists. It simply finesses the phase problem; however, the principles and procedures of diffraction analysis remain pertinent and are adopted in single-particle cryo-EM studies of biomolecules.

Chimeric Antigen Receptor T-cell (CAR-T) immunotherapies are dependent upon designed transmembrane proteins to bind target antigens and stimulate an immune response. The success or failure of these CARs is only partially predictable, yet recent work has highlighted the importance of antigen binding scFvs driving distinct oligomerization states with varied CAR-T efficacy. Here, we sought to determine the extracellular structure of the anti-CD19 CAR 47G4-CD828Z. Unexpectedly, the resolved crystal structure revealed an IgVL homodimer bound along an inverted VL|VL interface. We found that the VL-VH linker, designed to be cleavage resistant, was cleaved, and the VH and CAR hinge domains were absent from the crystal structure lattice. Molecular Dynamics simulations revealed that the inverted VL|VL interface was more stable than the canonical VL|VL configuration. Our work substantiates the need to interrogate the scFv structure and CAR oligomerization state for optimal CAR-T design.

Crystals of large macromolecular complexes often diffract quite poorly, typically having high solvent content, relatively feeble lattice contacts, quite weak subunit associations, and somewhat flexible interdomain linkages.Although resolution may be limited to dmin > 7-8 Å, the diffraction amplitudes should suffice, in principle, to specify conformational torsion angles; however, at such ultralow resolution, realizing and maintaining a suitable model within the radius of refinement convergence is a challenge.Important insights into biological processes may be obtained, but only if structural validity can be assured.

DNA double helices containing metal-mediated DNA (mmDNA) base pairs have been constructed from Ag+ and Hg2+ ions between pyrimidine:pyrimidine pairs with the promise of nanoelectronics. Rational design of mmDNA nanomaterials has been impractical without a complete lexical and structural description. Here, we explore the programmability of structural DNA nanotechnology toward its founding mission of self-assembling a diffraction platform for biomolecular structure determination. We employed the tensegrity triangle to build a comprehensive structural library of mmDNA pairs via X-ray diffraction and elucidated generalized design rules for mmDNA construction. We uncovered two binding modes: N3-dominant, centrosymmetric pairs and major groove binders driven by 5-position ring modifications. Energy gap calculations showed additional levels in the lowest unoccupied molecular orbitals (LUMO) of mmDNA structures, rendering them attractive molecular electronic candidates. This article is protected by copyright. All rights reserved.

Metal-mediated DNA (mmDNA) presents a pathway toward engineering bioinorganic and electronic behavior into DNA devices. Many chemical and biophysical forces drive the programmable chelation of metals between pyrimidine base pairs. Here, we developed a crystallographic method using the three-dimensional (3D) DNA tensegrity triangle motif to capture single- and multi-metal binding modes across granular changes to environmental pH using anomalous scattering. Leveraging this programmable crystal, we determined 28 biomolecular structures to capture mmDNA reactions. We found that silver(I) binds with increasing occupancy in T-T and U-U pairs at elevated pH levels, and we exploited this to capture silver(I) and mercury(II) within the same base pair and to isolate the titration points for homo- and heterometal base pair modes. We additionally determined the structure of a C-C pair with both silver(I) and mercury(II). Finally, we extend our paradigm to capture cadmium(II) in T-T pairs together with mercury(II) at high pH. The precision self-assembly of heterobimetallic DNA chemistry at the sub-nanometer scale will enable atomistic design frameworks for more elaborate mmDNA-based nanodevices and nanotechnologies.

Binding to the host cell receptors, CD4 and CCR5/CXCR4, triggers large-scale conformational changes in the HIV-1 envelope glycoprotein (Env) trimer [(gp120/gp41) 3 ] that promote virus entry into the cell. CD4-mimetic compounds (CD4mcs) comprise small organic molecules that bind in the highly conserved CD4-binding site of gp120 and prematurely induce inactivating Env conformational changes, including shedding of gp120 from the Env trimer. By inducing more “open,” antibody-susceptible Env conformations, CD4mcs also sensitize HIV-1 virions to neutralization by antibodies and infected cells to antibody-dependent cellular cytotoxicity (ADCC). Here, we report the design, synthesis, and evaluation of novel CD4mcs based on an indoline scaffold. Compared with our current lead indane scaffold CD4mc, BNM-III-170, several indoline CD4mcs exhibit increased potency and breadth against HIV-1 variants from different geographic clades. Viruses that were selected for resistance to the lead indane CD4mc, BNM-III-170, are susceptible to inhibition by the indoline CD4mcs. The indoline CD4mcs also potently sensitize HIV-1-infected cells to ADCC mediated by plasma from HIV-1-infected individuals. Crystal structures indicate that the indoline CD4mcs gain potency compared to the indane CD4mcs through more favorable π–π overlap from the indoline pose and by making favorable contacts with the vestibule of the CD4-binding pocket on gp120. The rational design of indoline CD4mcs thus holds promise for further improvements in antiviral activity, potentially contributing to efforts to treat and prevent HIV-1 infection.

Na+ is the most abundant ion in living organisms and plays essential roles in regulating nutrient uptake, muscle contraction, and neurotransmission. The identification of Na+ in protein structures is crucial for gaining a deeper understanding of protein function in a physiological context. LeuT, a bacterial homolog of the neurotransmitter:sodium symporter family, uses the Na+ gradient to power the uptake of amino acids into cells and has been used as a paradigm for the study of Na+-dependent transport systems. We have devised a low-energy multi-crystal approach for characterizing low-Z (Z ≤ 20) anomalous scattering ions such as Na+, Mg2+, K+, and Ca2+ by combining Bijvoet-difference Fourier syntheses for ion detection and f” refinements for ion speciation. Using the approach, we experimentally identify two Na+ bound near the central leucine binding site in LeuT. Using LeuT microcrystals, we also demonstrate that Na+ may be depleted to study conformational changes in the LeuT transport cycle.