Symmetric Homodimer Research Articles

Structural Basis for the Recognition of GPRC5D by Talquetamab, a Bispecific Antibody for Multiple Myeloma

Multiple myeloma (MM) is a complex hematological malignancy characterized by abnormal antibody production from plasma cells. Despite advances in the treatment, many patients experience disease relapse or become refractory to treatment. G-protein-coupled receptor class C group 5 member D (GPRC5D), an orphan GPCR predominantly expressed in MM cells, is emerging as a promising target for MM immunotherapy. Talquetamab, a Food and Drug Administration-approved T-cell-directing bispecific antibody developed for treatment of MM, targets GPRC5D. Here, we elucidate the structure of GPRC5D complexed with the Fab fragment of talquetamab, using cryo-electron microscopy, providing the basis for recognition of GPRC5D by the bispecific antibody. GPRC5D forms a symmetric homodimer with the interface between transmembrane helix (TM) 4 of one protomer and TM4/5 of the other protomer. A single talquetamab Fab interacts with the GPRC5D dimer with its orientation toward the dimer interface. All six complementarity-determining regions of talquetamab engage with extracellular loops and TM3/5/7. In particular, the side-chain of an arginine residue from the antibody penetrates into a shallow pocket on the extracellular surface of GPRC5D. The structure offers insights for optimizing antibody design against GPRC5D for relapsed or refractory MM therapy.

Oligomerization and a distinct tRNA-binding loop are important regulators of human arginyl-transferase function

The arginyl-transferase ATE1 is a tRNA-dependent enzyme that covalently attaches an arginine molecule to a protein substrate. Conserved from yeast to humans, ATE1 deficiency in mice correlates with defects in cardiovascular development and angiogenesis and results in embryonic lethality, while conditional knockouts exhibit reproductive, developmental, and neurological deficiencies. Despite the recent revelation of the tRNA binding mechanism and the catalytic cycle of yeast ATE1, the structure-function relationship of ATE1 in higher organisms is not well understood. In this study, we present the three-dimensional structure of human ATE1 in an apo-state and in complex with its tRNA cofactor and a peptide substrate. In contrast to its yeast counterpart, human ATE1 forms a symmetric homodimer, which dissociates upon binding of a substrate. Furthermore, human ATE1 includes a unique and extended loop that wraps around tRNAArg, creating extensive contacts with the T-arm of the tRNA cofactor. Substituting key residues identified in the substrate binding site of ATE1 abolishes enzymatic activity and results in the accumulation of ATE1 substrates in cells.

Assembly and activation of EBV latent membrane protein 1

Latent membrane protein 1 (LMP1) is the primary oncoprotein of Epstein-Barr virus (EBV) and plays versatile roles in the EBV life cycle and pathogenesis. Despite decades of extensive research, the molecular basis for LMP1 folding, assembly, and activation remains unclear. Here, we report cryo-electron microscopy structures of LMP1 in two unexpected assemblies: a symmetric homodimer and a higher-order filamentous oligomer. LMP1 adopts a non-canonical and unpredicted fold that supports the formation of a stable homodimer through tight and antiparallel intermolecular packing. LMP1 dimers further assemble side-by-side into higher-order filamentous oligomers, thereby allowing the accumulation and specific organization of the flexible cytoplasmic tails for efficient recruitment of downstream factors. Super-resolution microscopy and cellular functional assays demonstrate that mutations at both dimeric and oligomeric interfaces disrupt LMP1 higher-order assembly and block multiple LMP1-mediated signaling pathways. Our research provides a framework for understanding the mechanism of LMP1 and for developing potential therapies targeting EBV-associated diseases.

The molecular basis of tRNA selectivity by human pseudouridine synthase 3

Pseudouridine (Ψ), the isomer of uridine, is ubiquitously found in RNA, including tRNA, rRNA, and mRNA. Human pseudouridine synthase 3 (PUS3) catalyzes pseudouridylation of position 38/39 in tRNAs. However, the molecular mechanisms by which it recognizes its RNA targets and achieves site specificity remain elusive. Here, we determine single-particle cryo-EM structures of PUS3 in its apo form and bound to three tRNAs, showing how the symmetric PUS3 homodimer recognizes tRNAs and positions the target uridine next to its active site. Structure-guided and patient-derived mutations validate our structural findings in complementary biochemical assays. Furthermore, we deleted PUS1 and PUS3 in HEK293 cells and mapped transcriptome-wide Ψ sites by Pseudo-seq. Although PUS1-dependent sites were detectable in tRNA and mRNA, we found no evidence that human PUS3 modifies mRNAs. Our work provides the molecular basis for PUS3-mediated tRNA modification in humans and explains how its tRNA modification activity is linked to intellectual disabilities.

Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers

Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms. Apo polymerase protomers can adopt two drastically different conformations, which assemble into two distinct symmetric homodimers, that can themselves gather to form hexamers. Polymerase dimerization induces the stabilization of most polymerase domains, including the C-terminal domain that contributes the most to dimer’s interface, along with a lariat region that participates to the polymerase steadying. Binding to viral RNA induces significant conformational changes resulting in symmetric oligomer disruption and polymerase activation, suggesting the possible involvement of apo multimers as protecting systems that would stabilize the otherwise flexible C-terminal domains. Overall, these results provide insights into the multimerization capability of Hantavirus polymerase and may help to define antiviral compounds to counteract these life-threatening viruses.

Sequence differences between BAX and BAK core domains manifest as differences in their interactions with lipids.

The B-cell lymphoma 2 (BCL2) family members, BCL2-associated protein X (BAX) and BCL2 homologous antagonist killer (BAK), are required for programmed cell death via the mitochondrial pathway. When cells are stressed, damaged or redundant, the balance of power between the BCL2 family of proteins shifts towards BAX and BAK, allowing their transition from an inactive, monomeric state to a membrane-active oligomeric form that releases cytochrome c from the mitochondrial intermembrane space. That oligomeric state has an essential intermediate, a symmetric homodimer of BAX or BAK. Here we describe crystal structures of dimers of the core domain of BAX, comprising its helices α2-α5. These structures provide an atomic resolution description of the interactions that drive BAX homo-dimerisation and insights into potential interaction between core domain dimers and membrane lipids. The previously identified BAK lipid-interacting sites are not conserved with BAX and are likely to determine the differences between them in their interactions with lipids. We also describe structures of heterodimers of BAK/BAX core domains, yielding further insight into the differences in lipid binding between BAX and BAK.

Asymmetric pendrin homodimer reveals its molecular mechanism as anion exchanger

Pendrin (SLC26A4) is an anion exchanger expressed in the apical membranes of selected epithelia. Pendrin ablation causes Pendred syndrome, a genetic disorder associated with sensorineural hearing loss, hypothyroid goiter, and reduced blood pressure. However its molecular structure has remained unknown, limiting our understanding of the structural basis of transport. Here, we determine the cryo-electron microscopy structures of mouse pendrin with symmetric and asymmetric homodimer conformations. The asymmetric homodimer consists of one inward-facing protomer and the other outward-facing protomer, representing coincident uptake and secretion- a unique state of pendrin as an electroneutral exchanger. The multiple conformations presented here provide an inverted alternate-access mechanism for anion exchange. The structural and functional data presented here disclose the properties of an anion exchange cleft and help understand the importance of disease-associated variants, which will shed light on the pendrin exchange mechanism.

A Putative Receptor for Ferritin in Mollusks: Characterization of the Insulin-like Growth Factor Type 1 Receptor.

The ferritin secreted by mammals has been well documented, with the protein capable of localizing to cell membranes and facilitating the delivery of iron to cells through endocytosis. However, the presence of ferritin in the circulatory fluid of mollusks and its functions remain largely unknown. In this study, we aimed to investigate the potential interacting proteins of ferritin in the ark clam (SbFn) through the use of a pull-down assay. Our findings revealed the presence of an insulin-like growth factor type 1 receptor (IGF-1R) in ark clams, which was capable of binding to SbFn and was named SbIGF-1R. SbIGF-1R was found to be composed of two leucine-rich repeat domains (L domain), a cysteine-rich domain, three fibronectin type III domains, a transmembrane domain, and a tyrosine kinase domain. The ectodomain of SbIGF-1R was observed to form a symmetrical antiparallel homodimer in the shape of the letter 'A', with the fibronectin type III domains serving as its 'legs'. The mRNA expression of SbIGF-1R gene was detected ubiquitously in various tissues of the ark clam, with the highest expression levels found in hemocytes, as determined by qRT-PCR. Using a confocal microscopic and yeast two-hybrid assays, the interaction between SbIGF-1R and SbFn was further verified. The results showed that SbFn co-localized with SbIGF-1R on the cell membrane, and their interaction was expected to occur on the FNIII domains of the SbIGF-1R. In conclusion, our findings highlight the identification of a putative receptor, SbIGF-1R, for SbFn, demonstrating the versatility of IGF-1R in ark clams.

Structure and function analysis of Sam68 and hnRNP A1 synergy in the exclusion of exon 7 from SMN2 transcripts.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by the absence of a functional copy of the Survival of Motor Neuron 1 gene (SMN1). The nearly identical paralog, SMN2, cannot compensate for the loss of SMN1 because exon 7 is aberrantly skipped from most SMN2 transcripts, a process mediated by synergistic activities of Src-associated during mitosis, 68 kDa (Sam68/KHDRBS1) and heterogeneous nuclear ribonucleoprotein (hnRNP) A1. This results in the production of a truncated, nonfunctional protein that is rapidly degraded. Here, we present several crystal structures of Sam68 RNA-binding domain (RBD). Sam68-RBD forms stable symmetric homodimers by antiparallel association of helices α3 from two monomers. However, the details of domain organization and the dimerization interface differ significantly from previously characterized homologs. We demonstrate that Sam68 and hnRNP A1 can simultaneously bind proximal motifs within the central region of SMN2 (ex7). Furthermore, we show that the RNA-binding pockets of the two proteins are close to each other in their heterodimeric complex and identify contact residues using crosslinking-mass spectrometry. We present a model of the ternary Sam68·SMN2 (ex7)·hnRNP A1 complex that reconciles all available information on SMN1/2 splicing. Our findings have important implications for the etiology of SMA and open new avenues for the design of novel therapeutics to treat splicing diseases.

Structural insights into the PrpTA toxin-antitoxin system in Pseudoalteromonas rubra.

Bacteria could survive stresses by a poorly understood mechanism that contributes to the emergence of bacterial persisters exhibiting multidrug tolerance (MDT). Recently, Pseudoalteromonas rubra prpAT module was found to encode a toxin PrpT and corresponding cognate antidote PrpA. In this study, we first reported multiple individual and complex structures of PrpA and PrpT, which uncovered the high-resolution three-dimensional structure of the PrpT:PrpA2:PrpT heterotetramer with the aid of size exclusion chromatography-multi-angle light scattering experiments (SEC-MALS). PrpT:PrpA2:PrpT is composed of a PrpA homodimer and two PrpT monomers which are relatively isolated from each other and from ParE family. The superposition of antitoxin monomer structures from these structures highlighted the flexible C-terminal domain (CTD). A striking conformational change in the CTDs of PrpA homodimer depolymerized from homotetramer was provoked upon PrpT binding, which accounts for the unique PrpT-PrpARHH mutual interactions and further neutralizes the toxin PrpT. PrpA2-54-form I and II crystal structures both contain a doughnut-shaped hexadecamer formed by eight homodimers organized in a cogwheel-like form via inter-dimer interface dominated by salt bridges and hydrogen bonds. Moreover, PrpA tends to exist in solution as a homodimer other than a homotetramer (SEC-MALS) in the absence of flexible CTD. Multiple multi-dimers, tetramer and hexamer included, of PrpA2-54 mediated by the symmetric homodimer interface and the complicated inter-dimer interface could be observed in the solution. SEC-MALS assays highlighted that phosphate buffer (PB) and the increase in the concentration appear to be favorable for the PrpA2-54 oligomerization in the solution. Taken together with previous research, a model of PrpA2-54 homotetramer in complex with prpAT promoter and the improved mechanism underlying how PrpTA controls the plasmid replication were proposed here.

De novo design of protein homodimers containing tunable symmetric protein pockets

Function follows form in biology, and the binding of small molecules requires proteins with pockets that match the shape of the ligand. For design of binding to symmetric ligands, protein homo-oligomers with matching symmetry are advantageous as each protein subunit can make identical interactions with the ligand. Here, we describe a general approach to designing hyperstable C2 symmetric proteins with pockets of diverse size and shape. We first designed repeat proteins that sample a continuum of curvatures but have low helical rise, then docked these into C2 symmetric homodimers to generate an extensive range of C2 symmetric cavities. We used this approach to design thousands of C2 symmetric homodimers, and characterized 101 of them experimentally. Of these, the geometry of 31 were confirmed by small angle X-ray scattering and 2 were shown by crystallographic analyses to be in close agreement with the computational design models. These scaffolds provide a rich set of starting points for binding a wide range of C2 symmetric compounds.

Discovery of unusual dimeric piperazyl cyclopeptides encoded by a Lentzea flaviverrucosa DSM 44664 biosynthetic supercluster

Rare actinomycetes represent an underexploited source of new bioactive compounds. Here, we report the use of a targeted metabologenomic approach to identify piperazyl compounds in the rare actinomycete Lentzea flaviverrucosa DSM 44664. These efforts to identify molecules that incorporate piperazate building blocks resulted in the discovery and structural elucidation of two dimeric biaryl-cyclohexapeptides, petrichorins A and B. Petrichorin B is a symmetric homodimer similar to the known compound chloptosin, but petrichorin A is unique among known piperazyl cyclopeptides because it is an asymmetric heterodimer. Due to the structural complexity of petrichorin A, solving its structure required a combination of several standard chemical methods plus in silico modeling, strain mutagenesis, and solving the structure of its biosynthetic intermediate petrichorin C for confident assignment. Furthermore, we found that the piperazyl cyclopeptides comprising each half of the petrichorin A heterodimer are made via two distinct nonribosomal peptide synthetase (NRPS) assembly lines, and the responsible NRPS enzymes are encoded within a contiguous biosynthetic supercluster on the L. flaviverrucosa chromosome. Requiring promiscuous cytochrome p450 crosslinking events for asymmetric and symmetric biaryl production, petrichorins A and B exhibited potent in vitro activity against A2780 human ovarian cancer, HT1080 fibrosarcoma, PC3 human prostate cancer, and Jurkat human T lymphocyte cell lines with IC50 values at low nM levels. Cyclic piperazyl peptides and their crosslinked derivatives are interesting drug leads, and our findings highlight the potential for heterodimeric bicyclic peptides such as petrichorin A for inclusion in future pharmaceutical design and discovery programs.

Dimerization-dependent membrane tethering by Atg23 is essential for yeast autophagy.

SUMMARYEukaryotes maintain cellular health through the engulfment and subsequent degradation of intracellular cargo using macroautophagy. The function of Atg23, despite being critical to the efficiency of this process, is unclear due to a lack of biochemical investigations and an absence of any structural information. In this study, we use a combination of in vitro and in vivo methods to show that Atg23 exists primarily as a homodimer, a conformation facilitated by a putative amphipathic helix. We utilize small-angle X-ray scattering to monitor the overall shape of Atg23, revealing that it contains an extended rod-like structure spanning approximately 320 Å. We also demonstrate that Atg23 interacts with membranes directly, primarily through electrostatic interactions, and that these interactions lead to vesicle tethering. Finally, mutation of the hydrophobic face of the putative amphipathic helix completely precludes dimer formation, leading to severely impaired subcellular localization, vesicle tethering, Atg9 binding, and autophagic efficiency.

Functional Characterization of the Internal Symmetry of MRAP2 Antiparallel Homodimer.

The melanocortin receptors are defined as a series of vital pharmaceutical targets to regulate neuronal appetite and maintain controllable body weight for mammals and teleosts. Melanocortin receptor accessory protein 2 (MRAP2) functions as an essential accessory player that modulates the surface translocation and binding to a variety of endogenous or synthetic hormones of central melanocortin-4 receptor (MC4R) signaling. MRAP2 is a single-transmembrane protein and could form a functional symmetric antiparallel homodimer topology. Here, we inverted the N-terminal, transmembrane, and C-terminal domains and generated six distinct conformational variants of the mouse MRAP2 to explore the functional orientations and the internal symmetry of MRAP2 dimers. These remolded MRAP2 mutants showed proper assembly of the antiparallel homodimer and binding to the MC4R, but slightly altered the regulatory profile on the surface expression and the ligand-stimulated cAMP cascades of MC4R. This study elucidated the importance of the orientation of each domain of the single-transmembrane protein and revealed the pharmacological properties of the internal symmetry of the antiparallel homodimer for MRAP2.

TMEM120A contains a specific coenzyme A-binding site and might not mediate poking- or stretch-induced channel activities in cells.

TMEM120A, a member of the transmembrane protein 120 (TMEM120) family, has a pivotal function in adipocyte differentiation and metabolism, and may also contribute to sensing mechanical pain by functioning as an ion channel named TACAN. Here we report that expression of TMEM120A is not sufficient in mediating poking- or stretch-induced currents in cells and have solved cryo-electron microscopy (cryo-EM) structures of human TMEM120A (HsTMEM120A) in complex with an endogenous metabolic cofactor (coenzyme A, CoASH) and in the apo form. HsTMEM120A forms a symmetrical homodimer with each monomer containing an amino-terminal coiled-coil motif followed by a transmembrane domain with six membrane-spanning helices. Within the transmembrane domain, a CoASH molecule is hosted in a deep cavity and forms specific interactions with nearby amino acid residues. Mutation of a central tryptophan residue involved in binding CoASH dramatically reduced the binding affinity of HsTMEM120A with CoASH. HsTMEM120A exhibits distinct conformations at the states with or without CoASH bound. Our results suggest that TMEM120A may have alternative functional roles potentially involved in CoASH transport, sensing, or metabolism.

The Repressor C Protein, Pf4r, Controls Superinfection of Pseudomonas aeruginosa PAO1 by the Pf4 Filamentous Phage and Regulates Host Gene Expression.

It has been shown that the filamentous phage, Pf4, plays an important role in biofilm development, stress tolerance, genetic variant formation and virulence in Pseudomonas aeruginosa PAO1. These behaviours are linked to the appearance of superinfective phage variants. Here, we have investigated the molecular mechanism of superinfection as well as how the Pf4 phage can control host gene expression to modulate host behaviours. Pf4 exists as a prophage in PAO1 and encodes a homologue of the P2 phage repressor C and was recently named Pf4r. Through a combination of molecular techniques, ChIPseq and transcriptomic analyses, we show a critical site in repressor C (Pf4r) where a mutation in the site, 788799A>G (Ser4Pro), causes Pf4r to lose its function as the immunity factor against reinfection by Pf4. X-ray crystal structure analysis shows that Pf4r forms symmetric homo-dimers homologous to the E.coli bacteriophage P2 RepC protein. A mutation, Pf4r*, associated with the superinfective Pf4r variant, found at the dimer interface, suggests dimer formation may be disrupted, which derepresses phage replication. This is supported by multi-angle light scattering (MALS) analysis, where the Pf4r* protein only forms monomers. The loss of dimerisation also explains the loss of Pf4r’s immunity function. Phenotypic assays showed that Pf4r increased LasB activity and was also associated with a slight increase in the percentage of morphotypic variants. ChIPseq and transcriptomic analyses suggest that Pf4r also likely functions as a transcriptional regulator for other host genes. Collectively, these data suggest the mechanism by which filamentous phages play such an important role in P. aeruginosa biofilm development.

LZerD Protein-Protein Docking Webserver Enhanced With de novo Structure Prediction

Protein-protein docking is a useful tool for modeling the structures of protein complexes that have yet to be experimentally determined. Understanding the structures of protein complexes is a key component for formulating hypotheses in biophysics regarding the functional mechanisms of complexes. Protein-protein docking is an established technique for cases where the structures of the subunits have been determined. While the number of known structures deposited in the Protein Data Bank is increasing, there are still many cases where the structures of individual proteins that users want to dock are not determined yet. Here, we have integrated the AttentiveDist method for protein structure prediction into our LZerD webserver for protein-protein docking, which enables users to simply submit protein sequences and obtain full-complex atomic models, without having to supply any structure themselves. We have further extended the LZerD docking interface with a symmetrical homodimer mode. The LZerD server is available at https://lzerd.kiharalab.org/.

Two Intercalation Mechanisms of Oxazole Yellow Dimer (YOYO-1) into DNA.

The oxazole yellow dye, YOYO-1 (a symmetric homodimer), is a commonly used molecule for staining DNA. We applied the brightness analysis to study the intercalation of YOYO-1 into the DNA. We distinguished two binding modes of the dye to dsDNA: mono-intercalation and bis-intercalation. Bis-intercalation consists of two consecutive mono-intercalation steps, characterised by two distinct equilibrium constants (with the average number of base pair per binding site equals 3.5): and , respectively. Mono-intercalation dominates at high concentrations of YOYO-1. Bis-intercalation occurs at low concentrations.

Structure and mechanism of the human NHE1-CHP1 complex

Sodium/proton exchanger 1 (NHE1) is an electroneutral secondary active transporter present on the plasma membrane of most mammalian cells and plays critical roles in regulating intracellular pH and volume homeostasis. Calcineurin B-homologous protein 1 (CHP1) is an obligate binding partner that promotes NHE1 biosynthetic maturation, cell surface expression and pH-sensitivity. Dysfunctions of either protein are associated with neurological disorders. Here, we elucidate structures of the human NHE1-CHP1 complex in both inward- and inhibitor (cariporide)-bound outward-facing conformations. We find that NHE1 assembles as a symmetrical homodimer, with each subunit undergoing an elevator-like conformational change during cation exchange. The cryo-EM map reveals the binding site for the NHE1 inhibitor cariporide, illustrating how inhibitors block transport activity. The CHP1 molecule differentially associates with these two conformational states of each NHE1 monomer, and this association difference probably underlies the regulation of NHE1 pH-sensitivity by CHP1.

Dynamics and Infrared Spectrocopy of Monomeric and Dimeric Wild Type and Mutant Insulin.

The infrared spectroscopy and dynamics of -CO labels in wild type and mutant insulin monomer and dimer are characterized from molecular dynamics simulations using validated force fields. It is found that the spectroscopy of monomeric and dimeric forms in the region of the amide-I vibration differs for residues B24-B26 and D24-D26, which are involved in dimerization of the hormone. Also, the spectroscopic signatures change for mutations at position B24 from phenylalanine, which is conserved in many organisms and is known to play a central role in insulin aggregation, to alanine or glycine. Using three different methods to determine the frequency trajectories (solving the nuclear Schrödinger equation on an effective 1-dimensional potential energy curve, using instantaneous normal modes, and using parametrized frequency maps) leads to the same overall conclusions. The spectroscopic response of monomeric WT and mutant insulin differs from that of their respective dimers, and the spectroscopy of the two monomers in the dimer is also not identical. For the WT and F24A and F24G monomers, spectroscopic shifts are found to be ∼20 cm-1 for residues (B24-B26) located at the dimerization interface. Although the crystal structure of the dimer is that of a symmetric homodimer, dynamically the two monomers are not equivalent on the nanosecond time scale. Together with earlier work on the thermodynamic stability of the WT and the same mutants, it is concluded that combining computational and experimental infrared spectroscopy provides a potentially powerful way to characterize the aggregation state and dimerization energy of modified insulins.