Homodimeric Protein Research Articles

The prodomain of bone morphogenetic protein 2 promotes dimerization and cleavage of BMP6 homodimers and BMP2/6 heterodimers

Bone morphogenetic protein 2 (BMP2) and BMP6 are key regulators of systemic iron homeostasis. All BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments, but nothing is known about how BMP2 or BMP6 homodimeric or heterodimeric precursor proteins are proteolytically activated. Here, we conducted in vitro cleavage assays, which revealed that BMP2 is sequentially cleaved by furin at two sites, initially at a site upstream of the mature ligand, and then at a site adjacent to the ligand domain, while BMP6 is cleaved at a single furin motif. Cleavage of both sites of BMP2 is required to generate fully active BMP2 homodimers when expressed in Xenopus embryos or liver endothelial cells, and fully active BMP2/6 heterodimers in Xenopus. We analyzed BMP activity in Xenopus embryos expressing chimeric proteins consisting of the BMP2 prodomain and BMP6 ligand domain, or vice versa. We show that the prodomain of BMP2 is necessary and sufficient to generate active BMP6 homodimers and BMP2/6 heterodimers, whereas the BMP6 prodomain cannot generate active BMP2 homodimers or BMP2/6 heterodimers. We examined BMP2 and BMP6 homodimeric and heterodimeric ligands generated from native and chimeric precursor proteins expressed in Xenopus embryos. Whereas native BMP6 is not cleaved when expressed alone, it is cleaved to generate BMP2/6 heterodimers when co-expressed with BMP2. Furthermore, BMP2-6 chimeras are cleaved to generate BMP6 homodimers. Our findings reveal an important role for the BMP2 prodomain in dimerization and proteolytic activation of BMP6.

Broad-spectrum coronavirus neutralization induced by hetero RBD-Fc protein vaccine.

In the landscape of infectious diseases, human coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2 pose significant threats, characterized by severe respiratory illnesses and notable resistance to conventional treatments due to their rapid evolution and the emergence of diverse variants, particularly within SARS-CoV-2. This study investigated the development of broad-spectrum coronavirus vaccines using heterodimeric RBD-Fc proteins engineered through the "Knob-into-Hole" technique. We constructed various recombinant proteins incorporating the receptor-binding domains (RBDs) of different coronaviruses. Heterodimers combining RBDs from SARS-CoV-2 with those of SARS-CoV or MERS-CoV elicited superior neutralizing responses compared to homodimeric proteins in murine models. Additionally, heterotetrameric proteins, specifically D614G_Delta/BA.1_XBB.1.5-RBD and MERS_D614G/BA.1_XBB.1.5-RBD, elicited remarkable breadth and potency in neutralizing all known SARS-CoV-2 variants, SARS-CoV, related sarbecoviruses like GD-Pangolin and WIV1, and even MERS-CoV pseudoviruses. Furthermore, these heterotetrameric proteins also demonstrated enhanced cellular immune responses. These findings underscore the potential of recombinant hetero proteins as a universal vaccine strategy against current and future coronavirus threats.

The Role of Mitochondrial Pyruvate Carrier in Neurological Disorders.

The mitochondrial pyruvate carrier (MPC) is a specific protein complex located in the inner mitochondrial membrane. Comprising a heterodimer of two homodimeric membrane proteins, mitochondrial pyruvate carrier 1 and mitochondrial pyruvate carrier 2, MPC connects cytoplasmic metabolism to mitochondrial metabolism by transferring pyruvate from the cytoplasm to the mitochondria. The nervous system requires substantial energy to maintain its function, and the mitochondrial energy supply is closely linked to neurological function. Mitochondrial dysfunction can induce or exacerbate intracerebral pathologies. MPC influences mitochondrial function due to its specific role as a pyruvate transporter. However, recent studies on MPC and mitochondrial dysfunction in neurological disorders have yielded controversial results, and the underlying mechanisms remain unclear. In this brief review, we provide an overview of the structure and function of MPC. We further discuss the potential mechanisms and feasibility of targeting MPC in treating Parkinson's disease, Alzheimer's disease, and cerebral ischemia/hypoxia injury. This review aims to offer insights into MPC as a target for clinical treatment.

Biophysical and structural studies of fibulin-2

Fibulin-2 is a multidomain, disulfide-rich, homodimeric protein which belongs to a broader extracellular matrix family. It plays an important role in the development of elastic fiber structures. Malfunction of fibulin due to mutation or poor expression can result in a variety of diseases including synpolydactyly, limb abnormalities, eye disorders leading to blindness, cardiovascular diseases and cancer. Traditionally, fibulins have either been produced in mammalian cell systems or were isolated from the extracellular matrix, a procedure that results in poor availability for structural and functional studies. Here, we produced seven fibulin-2 constructs covering 62% of the mature protein (749 out of 1195 residues) using a prokaryotic expression system. Biophysical studies confirm that the purified constructs are folded and that the presence of disulfide bonds within the constructs makes them extremely thermostable. In addition, we solved the first crystal structure for any fibulin isoform, a structure corresponding to the previously suggested three motifs related to anaphylatoxin. The structure reveals that the three anaphylatoxins moieties form a single-domain structure.

The BMP2 prodomain promotes dimerization and cleavage of BMP6 homodimers and BMP2/6 heterodimers in vivo.

Bone morphogenetic protein 2 (BMP2) and BMP6 are key regulators of systemic iron homeostasis. All BMPs are generated as inactive precursor proteins that dimerize and are cleaved to generate the bioactive ligand and inactive prodomain fragments, but nothing is known about how BMP2 or BMP6 homodimeric or heterodimeric precursor proteins are proteolytically activated. Here, we conducted in vitro cleavage assays, which revealed that BMP2 is sequentially cleaved by furin at two sites, initially at a site upstream of the mature ligand, and then at a site adjacent to the ligand domain, while BMP6 is cleaved at a single furin motif. Cleavage of both sites of BMP2 is required to generate fully active BMP2 homodimers when expressed in Xenopus embryos or liver endothelial cells, and fully active BMP2/6 heterodimers in Xenopus . We analyzed BMP activity in Xenopus embryos expressing chimeric proteins consisting of the BMP2 prodomain and BMP6 ligand domain, or vice versa. We show that the prodomain of BMP2 is necessary and sufficient to generate active BMP6 homodimers and BMP2/6 heterodimers, whereas the BMP6 prodomain cannot generate active BMP2 homodimers or BMP2/6 heterodimers. We examined BMP2 and BMP6 homodimeric and heterodimeric ligands generated from native and chimeric precursor proteins expressed in Xenopus embryos. Whereas native BMP6 is not cleaved when expressed alone, it is cleaved to generate BMP2/6 heterodimers when co-expressed with BMP2. Furthermore, BMP2-6 chimeras are cleaved to generate BMP6 homodimers. Our findings reveal an important role for the BMP2 prodomain in dimerization and proteolytic activation of BMP6.

Labeling of methyl groups: a streamlined protocol and guidance for the selection of 2H precursors based on molecular weight.

A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2O and 2H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D2O) or deuterated glucose is not required to obtain 1H-13C correlated NMR spectra of a 50kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100-150kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the β-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.

Structural dynamics of the heme pocket and intersubunit coupling in the dimeric hemoglobin from Scapharca inaequivalvis.

Cooperativity is essential for the proper functioning of numerous proteins by allosteric interactions. Hemoglobin from Scapharca inaequivalvis (HbI) is a homodimeric protein that can serve as a minimal unit for studying cooperativity. We investigated the structural changes in HbI after carbon monoxide dissociation using time-resolved resonance Raman spectroscopy and observed structural rearrangements in the Fe-proximal histidine bond, the position of the heme in the pocket, and the hydrogen bonds between heme and interfacial water upon ligand dissociation. Some of the spectral changes were different from those observed for human adult hemoglobin due to differences in subunit assembly and quaternary changes. The structural rearrangements were similar for the singly and doubly dissociated species but occurred at different rates. The rates of the observed rearrangements indicated that they occurred synchronously with subunit rotation and are influenced by intersubunit coupling, which underlies the positive cooperativity of HbI.

Deciphering S100B Allosteric Signaling: The Role of a Peptide Target, TRTK-12, as an Ensemble Modulator.

Allostery is an essential biological phenomenon in which perturbation at one site in a biomolecule elicits a functional response at a distal location(s). It is integral to biological processes, such as cellular signaling, metabolism, and transcription regulation. Understanding allostery is also crucial for rational drug discovery. In this work, we focus on an allosteric S100B protein that belongs to the S100 class of EF-hand Ca2+-binding proteins. The Ca2+-binding affinity of S100B is modulated allosterically by TRTK-12 peptide binding 25 Å away from the Ca2+-binding site. We investigated S100B allostery by carrying out nuclear magnetic resonance (NMR) measurements along with microsecond-long molecular dynamics (MD) simulations on S100B/Ca2+ with/without TRTK-12 at different NaCl salt concentrations. NMR HSQC results show that TRTK-12 reorganizes how S100B/Ca2+ responds to different salt concentrations at both orthosteric and allosteric sites. The MD data suggest that TRTK-12 breaks the dynamic aromatic and hydrogen-bond interactions (not observed in X-ray crystallographic structures) between the hinge/helix and Ca2+-binding EF-hand loop of the two subunits in the homodimeric protein. This triggers rearrangement in the protein network architectures and leads to allosteric communication. Finally, computational studies of S100B at distinct ionic strengths suggest that ligand-bound species are more robust to the changing environment relative to the S100B/Ca2+ complex.

Characterization of the iron-sulfur clusters in the nitrogenase-like reductase CfbC/D required for coenzyme F430 biosynthesis.

Coenzyme F430 is a nickel-containing tetrapyrrole, serving as the prosthetic group of methyl-coenzyme M reductase in methanogenic and methanotrophic archaea. During coenzyme F430 biosynthesis, the tetrapyrrole macrocycle is reduced by the nitrogenase-like CfbC/D system consisting of the reductase component CfbC and the catalytic component CfbD. Both components are homodimeric proteins, each carrying a [4Fe-4S] cluster. Here, the ligands of the [4Fe-4S] clusters of CfbC2 and CfbD2 were identified revealing an all cysteine ligation of both clusters. Moreover, the midpoint potentials of the [4Fe-4S] clusters were determined to be -256 mV for CfbC2 and -407 mV for CfbD2. These midpoint potentials indicate that the consecutive thermodynamically unfavorable 6 individual "up-hill" electron transfers to the organic moiety of the Ni2+-sirohydrochlorin a,c-diamide substrate require an intricate interplay of ATP-binding, hydrolysis, protein complex formation and release to drive product formation, which is a common theme in nitrogenase-like systems.

Probing the active site of Class 3 L-asparaginase by mutagenesis. I. Tinkering with the zinc coordination site of ReAV.

ReAV, the inducible Class-3 L-asparaginase from the nitrogen-fixing symbiotic bacterium Rhizobium etli, is an interesting candidate for optimizing its enzymatic potential for antileukemic applications. Since it has no structural similarity to known enzymes with this activity, it may offer completely new ways of approach. Also, as an unrelated protein, it would evade the immunological response elicited by other asparaginases. The crystal structure of ReAV revealed a uniquely assembled protein homodimer with a highly specific C135/K138/C189 zinc binding site in each subunit. It was also shown before that the Zn2+ cation at low and optimal concentration boosts the ReAV activity and improves substrate specificity, which indicates its role in substrate recognition. However, the detailed catalytic mechanism of ReAV is still unknown. In this work, we have applied site-directed mutagenesis coupled with enzymatic assays and X-ray structural analysis to elucidate the role of the residues in the zinc coordination sphere in catalysis. Almost all of the seven ReAV muteins created in this campaign lost the ability to hydrolyze L-asparagine, confirming our predictions about the significance of the selected residues in substrate hydrolysis. We were able to crystallize five of the ReAV mutants and solve their crystal structures, revealing some intriguing changes in the active site area as a result of the mutations. With alanine substitutions of Cys135 or Cys189, the zinc coordination site fell apart and the mutants were unable to bind the Zn2+ cation. Moreover, the absence of Lys138 induced atomic shifts and conformational changes of the neighboring residues from two active-site Ser-Lys tandems. Ser48 from one of the tandems, which is hypothesized to be the catalytic nucleophile, usually changes its hydration pattern in response to the mutations. Taken together, the results provide many useful clues about the catalytic mechanism of the enzyme, allowing one to cautiously postulate a possible enzymatic scenario.

Bacteria at Work - Experimental and Theoretical Studies Reveal the Catalytic Mechanism of Ectoine Synthase.

Ectoine synthase (EctC) catalyses the ultimate step of ectoine biosynthesis, a kosmotropic compound produced as compatible solute by many bacteria and some archaea or eukaryotes. EctC is an Fe2+-dependent homodimeric cytoplasmic protein. Using Mössbauer spectroscopy, molecular dynamics simulations and QM/MM calculations, we determined the most likely coordination number and geometry of the Fe2+ ion and proposed a mechanism of the EctC-catalysed reaction. Most notably, we show that apart from the three amino acids binding to the iron ion (Glu57, Tyr84 and His92), one water molecule and one hydroxide ion are required as additional ligands for the reaction to occur. They fill the first coordination sphere of the Fe2+-cofactor and act as critical proton donors and acceptors during the cyclization reaction.

Identification and characterization of a novel type of ketohexokinase from the haloarchaeon Haloferax volcanii.

Ketohexokinase (KHK) catalyzes the ATP-dependent phosphorylation of fructose, forming fructose-1-phosphate and ADP. The enzyme is well studied in Eukarya, in particular in humans and other vertebrates, but homologs have not been identified in Bacteria and Archaea. Here we report the identification of a novel type of KHK from the haloarchaeon Haloferax volcanii (HvKHK). The encoding gene khk was identified as HVO_1812. The gene was expressed as a 90-kDa homodimeric protein, catalyzing the phosphorylation of fructose with a Vmax value of 59 U/mg and apparent KM values for ATP and fructose of 0.47 and 1.29 mM, respectively. Homologs of HvKHK were only identified in a few haloarchaea and halophilic Bacteria. The protein showed low sequence identity to characterized KHKs from Eukarya and phylogenetic analyses indicate that haloarchaeal KHKs are largely separated from eukaryal KHKs. This is the first report of the identification of KHKs in prokaryotes that form a novel cluster of sugar kinases within the ribokinase/pfkB superfamily.

Binding of a single nitric oxide molecule is sufficient to disrupt DNA binding of the nitrosative stress regulator NsrR.

The regulatory protein NsrR, a member of the Rrf2 protein superfamily, plays a major role in the cellular response to nitrosative stress in many benign and pathogenic bacteria. The homodimeric protein binds a [4Fe-4S] cluster in each subunit (termed holo NsrR), and represses transcription of genes primarily involved in NO detoxification. Holo NsrR reacts rapidly with multiple NO molecules per [4Fe-4S] cluster, via a complex reaction, with loss of DNA binding and formation of NsrR-bound iron-nitrosyl species. However, the point at which DNA binding is lost is unknown. Here, we demonstrate using surface plasmon resonance (SPR) and native mass spectrometry (MS) that holo NsrR binds the promoter regions of NsrR-regulated genes with promoter-dependent nanomolar affinity, while hemi-apo NsrR (i.e. one cluster per dimer) binds >10-fold less tightly, and the cluster-free (apo) form not at all. Strikingly, native MS provided detailed information about the reaction of NO with the physiologically relevant form of NsrR, i.e. DNA-bound dimeric NsrR. Reaction with a single NO molecule per NsrR dimer is sufficient to abolish DNA binding. This exquisite sensitivity of DNA binding to NO is consistent with the importance of de-repressing NO detoxification systems at the earliest opportunity to minimise damage due to nitrosative stress. Furthermore, the data show that previously characterised iron-nitrosyls, which form at higher ratios of NO to [4Fe-4S], are not physiologically relevant for regulating the NsrR on/off switch.

Small Molecule Inhibitors Targeting the "Undruggable" Survivin: The Past, Present, and Future from a Medicinal Chemist's Perspective.

Survivin, a homodimeric protein and a member of the IAP family, plays a vital function in cell survival and cycle progression by interacting with various proteins and complexes. Its expression is upregulated in cancers but not detectable in normal tissues. Thus, it has been regarded and validated as an ideal cancer target. However, survivin is "undruggable" due to its lack of enzymatic activities or active sites for small molecules to bind/inhibit. Academic and industrial laboratories have explored different strategies to overcome this hurdle over the past two decades, with some compounds advanced into clinical testing. These strategies include inhibiting survivin expression, its interaction with binding partners and homodimerization. Here, we provide comprehensive analyses of these strategies and perspective on different small molecule survivin inhibitors to help drug discovery targeting "undruggable" proteins in general and survivin specifically with a true survivin inhibitor that will prevail in the foreseeable future.

A Kinetic Transition Network Model Reveals the Diversity of Protein Dimer Formation Mechanisms.

Protein homodimers have been classified as three-state or two-state dimers depending on whether a folded monomer forms before association, but the details of the folding-binding mechanisms are poorly understood. Kinetic transition networks of conformational states have provided insight into the folding mechanisms of monomeric proteins, but extending such a network to two protein chains is challenging as all the relative positions and orientations of the chains need to be included, greatly increasing the number of degrees of freedom. Here, we present a simplification of the problem by grouping all states of the two chains into two layers: a dissociated and an associated layer. We combined our two-layer approach with the Wako-Saito-Muñoz-Eaton method and used Transition Path Theory to investigate the dimer formation kinetics of eight homodimers. The analysis reveals a remarkable diversity of dimer formation mechanisms. Induced folding, conformational selection, and rigid docking are often simultaneously at work, and their contribution depends on the protein concentration. Pre-folded structural elements are always present at the moment of association, and asymmetric binding mechanisms are common. Our two-layer network approach can be combined with various methods that generate discrete states, yielding new insights into the kinetics and pathways of flexible binding processes.

Mycobacterium tuberculosis Ku Stimulates Multi-round DNA Unwinding by UvrD1 Monomers

Mycobacterium tuberculosis is the causative agent of Tuberculosis. During the host response to infection, the bacterium is exposed to both reactive oxygen species and nitrogen intermediates that can cause DNA damage. It is becoming clear that the DNA damage response in Mtb and related actinobacteria function via distinct pathways as compared to well-studied model bacteria. For example, we have previously shown that the DNA repair helicase UvrD1 is activated for processive unwinding via redox-dependent dimerization. In addition, mycobacteria contain a homo-dimeric Ku protein, homologous to the eukaryotic Ku70/Ku80 dimer, that plays roles in double-stranded break repair via non-homologous end-joining. Kuhas been shown to stimulate the helicase activity of UvrD1, but the molecular mechanism, as well as which redox form of UvrD1 is activated, is unknown. We show here that Ku specifically stimulates multi-round unwinding by UvrD1 monomers which are able to slowly unwind DNA, but at rates 100-fold slower than the dimer. We also demonstrate that the UvrD1 C-terminal Tudor domain is required for the formation of a Ku-UvrD1 protein complex and activation. We show that Mtb Ku dimers bind with high nearest neighbor cooperativity to duplex DNA and that UvrD1 activation is observed when the DNA substrate is bound with two or three Ku dimers. Our observations reveal aspects of the interactions between DNA, Mtb Ku, and UvrD1 and highlight the potential role of UvrD1 in multiple DNA repair pathways through different mechanisms of activation.

A novel SARS-CoV-2 Beta RBD DNA vaccine directly targeted to antigen-presenting cells induces strong humoral and T cell responses

Throughout the COVID-19 pandemic, several variants of concern (VoC) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have evolved, affecting the efficacy of the approved COVID-19 vaccines. To address the need for vaccines that induce strong and persistent cross-reactive neutralizing antibodies and T cell responses, we developed a prophylactic SARS-CoV-2 vaccine candidate based on our easily and rapidly adaptable plasmid DNA vaccine platform. The vaccine candidate, referred to here as VB2129, encodes a protein homodimer consisting of the receptor binding domain (RBD) from lineage B.1.351 (Beta) of SARS-CoV-2, a VoC with a severe immune profile, linked to a targeting unit (human LD78β/CCL3L1) that binds chemokine receptors on antigen-presenting cells (APCs) and a dimerization unit (derived from the hinge and CH3 exons of human IgG3). Immunogenicity studies in mice demonstrated that the APC-targeted vaccine induced strong antibody responses to both homologous Beta RBD and heterologous RBDs derived from Wuhan, Alpha, Gamma, Delta, and Omicron BA.1 variants, as well as cross-neutralizing antibodies against these VoC. Overall, preclinical data justify the exploration of VB2129 as a potential booster vaccine that induces broader antibody- and T cell-based protection against current and future SARS-CoV-2 VoC.

Protein dynamics govern the oxyferrous state lifetime of an artificial oxygen transport protein

It has long been known that the alteration of protein side chains that occlude or expose the heme cofactor to water can greatly affect the stability of the oxyferrous heme state. Here, we demonstrate that the rate of dynamically driven water penetration into the core of an artificial oxygen transport protein also correlates with oxyferrous state lifetime by reducing global dynamics, without altering the structure of the active site, via the simple linking of the two monomers in a homodimeric artificial oxygen transport protein using a glycine-rich loop. The tethering of these two helices does not significantly affect the active site structure, pentacoordinate heme-binding affinity, reduction potential, or gaseous ligand affinity. It does, however, significantly reduce the hydration of the protein core, as demonstrated by resonance Raman spectroscopy, backbone amide hydrogen exchange, and pKa shifts in buried histidine side chains. This further destabilizes the charge-buried entatic state and nearly triples the oxyferrous state lifetime. These data are the first direct evidence that dynamically driven water penetration is a rate-limiting step in the oxidation of these complexes. It furthermore demonstrates that structural rigidity that limits water penetration is a critical design feature in metalloenzyme construction and provides an explanation for both the failures and successes of earlier attempts to create oxygen-binding proteins.

FRI001 Tanycytic Thyroid-stimulating Hormone Receptor Activation Increases Blood-Hypothalamus Barrier Permeability And Induces Appetite In Female Mice

Abstract Disclosure: H.S. Kannangara: None. B. Jocoll: None. S. Miyashita: None. A. Gumerova: None. F. Korkmaz: None. R. Witztum: None. S. Sims: None. U. Cheliadinova: None. G. Pevnev: None. O. Moldavski: None. T. Frolinger: None. T. Yuen: None. V. Ryu: None. M. Zaidi: None. In addition to its well-known action to trigger thyroid hormone production, thyroid-stimulating hormone (TSH) is released in the brain, and its receptor (TSHR) has been found in brain sites, including the tanycytes of the third ventricle, that regulate ingestive behavior and convey metabolic information across the blood-hypothalamus barrier (BHB). Our recent work highlights largely undiscovered role for TSHRs in the tanycytes. Here, we tested the hypothesis that tanycytic TSHR activation triggers rapid remodeling of BHB vascular permeability inducing appetite. We compared hypothalamic vascular permeability of food deprived (FD) and ad libitum fed (AL) Tshr+/- and their wild type (WT) counterparts exploiting transmission electron microscopy studies and immunofluorescence for constitutive tight junction protein Claudin-1 and homodimer glycoprotein MECA-32, primarily expressed in tanycytes and endothelial cells, respectively. Fasting significantly increased the organization of tanycytic honeycomb pattern of Claudin-1 and fenestral MECA-32-associated diaphragms in the median eminence and arcuate nucleus implying decreased BHB vascular permeability in Tshr+/- mice. Transmission electron microscopy confirmed immunofluorescence results. In a separate gain-of-function experiment, TSHR activation by the small molecule MS438 increased BHB vascular permeability as evidenced by decreased Claudin-1 and MECA-32 immunostaining. Tanycytic TSHR activation significantly increased both food foraging and intake as during one-hour refeeding after FD. Surprisingly, refeeding restored neuronal activation marker c-Fos expression in the paraventricular nucleus of the hypothalamus, parabrachial nucleus and nucleus of the solitary tract, though failed to do so in the paraventricular nucleus and nucleus of the solitary tract of MS438-injected mice. Collectively, these data infer that modulation of tanycytic TSHRs plays an important role in BHB plasticity in relation with ingestive behavior. Presentation: Friday, June 16, 2023

Cryo-EM structure of the KLHL22 E3 ligase bound to an oligomeric metabolic enzyme

CULLIN-RING ligases constitute the largest group of E3 ubiquitin ligases. While some CULLIN family members recruit adapters before engaging further with different substrate receptors, homo-dimeric BTB-Kelch family proteins combine adapter and substrate receptor into a single polypeptide for the CULLIN3 family. However, the entire structural assembly and molecular details have not been elucidated to date. Here, we present a cryo-EM structure of the CULLIN3RBX1 in complex with Kelch-like protein 22 (KLHL22) and a mitochondrial glutamate dehydrogenase complex I (GDH1) at 3.06Å resolution. The structure adopts a W-shaped architecture formed by E3 ligase dimers. Three CULLIN3KLHL22-RBX1 dimers were found to bedynamically associated with a single GDH1 hexamer. CULLIN3KLHL22-RBX1 ligase mediated the polyubiquitination of GDH1 invitro. Together, these results enabled the establishment of a structural model for understanding the complete assembly of BTB-Kelch proteins with CULLIN3 and how together they recognize oligomeric substrates and target them for ubiquitination.