Intersubunit Interactions Research Articles

A critical inter-subunit interaction for the transmission of the allosteric signal in the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase is a key regulatory enzyme involved in starch and glycogen synthesis in plants and bacteria, respectively. It has been hypothesized that inter-subunit communications are important for the allosteric effect in this enzyme. However, no specific interactions have been identified as part of the regulatory signal. The enzyme from Agrobacterium tumefaciens is a homotetramer allosterically regulated by fructose 6-phosphate and pyruvate. Three pairs of distinct subunit-subunit interfaces are present. Here we focus on an interface that features two symmetrical interactions between Arg11 and Asp141 from one subunit with residues Asp141 and Arg11 of the neighbor subunit, respectively. Previously, scanning mutagenesis showed that a mutation at the Arg11 position disrupted the activation of the enzyme. Considering the distance of these residues from the allosteric and catalytic sites, we hypothesized that the interaction between Arg11 and Asp141 is critical for allosteric signaling rather than effector binding. To prove our hypothesis, we mutated those two sites (D141A, D141E, D141N, D141R, R11D, and R11K) and performed kinetic and binding analysis. Mutations that altered the charge affected the regulation the most. To prove that the interaction per se (rather than the presence of specific residues) is critical, we partially rescued the R11D protein by introducing a second mutation (R11D/D141R). This could not restore the activator effect on kcat , but it did rescue the effect on substrate affinity. Our results indicate the critical functional role of Arg11 and Asp141 to relay the allosteric signal in this subunit interface.

DELE1 oligomerization promotes integrated stress response activation.

Mitochondria are dynamic organelles that continually respond to cellular stress. Recent studies have demonstrated that mitochondrial stress is relayed from mitochondria to the cytosol by the release of a proteolytic fragment of DELE1 that binds to the eIF2α kinase HRI to initiate integrated stress response (ISR) signaling. We report the cryo-electron microscopy structure of the C-terminal cleavage product of human DELE1, which assembles into a high-order oligomer. The oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization, and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. We further show that assembly-impaired DELE1 mutants are compromised in their ability to induce HRI-dependent ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.

Effect of Colchicine Binding Site Inhibitors on the Tubulin Intersubunit Interaction.

Microtubules are dynamic, non-covalent polymers consisting of α- and β-tubulin subunits that are involved in a wide range of intracellular processes. The polymerization and dynamics of microtubules are regulated by many factors, including small molecules that interact with different sites on the tubulin dimer. Colchicine binding site inhibitors (CBSIs) destabilize microtubules and inhibit tubulin polymerization, leading to cell cycle arrest. Because of their therapeutic potential, the molecular mechanism of CBSI function is an area of active research. Nevertheless, important details of this mechanism have yet to be resolved. In this study, we use atomistic molecular dynamics simulations to show that the binding of CBSIs to the tubulin heterodimer leads to the weakening of tubulin intersubunit interaction. Using atomistic molecular dynamics simulations and binding free energy calculations, we show that CBSIs act as protein-protein interaction inhibitors and destabilize interlinkage between α and β subunits, which is crucial for longitudinal contacts in the microtubule lattice. Our results offer new insight into the mechanisms of microtubule polymerization inhibition by colchicine and its analogs.

Structural insights into BCDX2 complex function in homologous recombination.

Homologous recombination (HR) fulfils a pivotal role in the repair of DNA double-strand breaks and collapsed replication forks1. HR depends on the products of several paralogues of RAD51, including the tetrameric complex of RAD51B, RAD51C, RAD51D and XRCC2 (BCDX2)2. BCDX2 functions as a mediator of nucleoprotein filament assembly byRAD51andsingle-stranded DNA (ssDNA) during HR, but its mechanism remains undefined. Here we report cryogenic electron microscopy reconstructions of human BCDX2 in apo and ssDNA-bound states. The structures reveal how the amino-terminal domains of RAD51B, RAD51C and RAD51D participate in inter-subunit interactions that underpin complex formation and ssDNA-binding specificity. Single-molecule DNA curtain analysis yields insights into how BCDX2 enhances RAD51-ssDNA nucleoprotein filament assembly. Moreover, our cryogenic electron microscopy and functional analyses explain how RAD51C alterations found in patients with cancer3-6 inactivate DNA binding and theHR mediator activity of BCDX2. Our findings shed light on the role of BCDX2 in HR and provide a foundation for understanding how pathogenic alterations in BCDX2 impact genome repair.

Site-specific labeling of SBDS to monitor interactions with the 60S ribosomal subunit.

The Shwachman-Diamond syndrome (SDS) is a rare inherited ribosomopathy that is predominantly caused by mutations in the Shwachman-Bodian-Diamond Syndrome gene (SBDS). SBDS is a ribosomal maturation factor that is essential for the release of eukaryotic translation initiation factor 6 (eIF6) from 60S ribosomal subunits during the late stages of 60S maturation. Release of eIF6 is critical to permit inter-subunit interactions between the 60S and 40S subunits and to form translationally competent 80S monosomes. SBDS has three key domains that are highly flexible and adopt varied conformations in solution. To better understand the domain dynamics of SBDS upon binding to 60S and to assess the effects of SDS-disease specific mutations, we aimed to site-specifically label individual domains of SBDS. Here we detail the generation of a fluorescently labeled SBDS to monitor the dynamics of select domains upon binding to 60S. We describe the incorporation of 4-azido-l-phenylalanine (4AZP), a noncanonical amino acid in human SBDS. Site-specific labeling of SBDS using fluorophore and assessment of 60S binding activity are also described. Such labeling approaches to capture the interactions of individual domains of SBDS with 60S are also applicable to study the dynamics of other multi-domain proteins that interact with the ribosomal subunits.

Molecular mechanism and energetics of coupling between substrate binding and product release in the F1-ATPase catalytic cycle

F1-ATPase is a motor protein that couples the rotation of its rotary [Formula: see text] subunit with ATP synthesis or hydrolysis. Single-molecule experiments indicate that nucleotide binding and release events occur almost simultaneously during the synthesis cycle, allowing the energy gain due to spontaneous binding of ADP to one catalytic [Formula: see text] subunit to be directly harnessed for driving the release of ATP from another rather than being dissipated as heat. Here, we examine the unknown mechanism of this coupling that is critical for an exceptionally high mechanochemical efficiency of F1-ATPase by means of all-atom free-energy simulations. We find that nondissipative and kinetically fast progression of the motor in the synthesis direction requires a concerted conformational change involving the closure of the ADP-binding [Formula: see text] subunit followed by the gradual opening of the ATP-releasing [Formula: see text] subunit over the course of the 30 to 40° rotary substep of the [Formula: see text] subunit. This rotary substep, preceding the ATP-dependent metastable state, allows for the recovery of a large portion of the ADP binding energy in the conformation of ATP-bound [Formula: see text] that gradually adopts the low-affinity conformation, captured also by the recent cryo-EM structure of this elusive state. The release of ATP from this nearly open conformation leads to its further opening, which enables the progression of the motor to the next catalytic metastable state. Our simulations explain this energy conversion mechanism in terms of intersubunit and ligand-protein interactions.

DELE1 oligomers revealed by cryo-EM structure promote the integrated stress response activation.

Mitochondria are dynamic organelles that must continually adapt and respond to cellular stress, and perturbations in mitochondrial function trigger the integrated stress response (ISR). Recent studies demonstrated that mitochondrial stress can be relayed from the mitochondrial interior to the cytosol by the release of a C-terminal proteolytic fragment of DELE1 that binds to the HRI kinase to initiate integrate stress response signaling. Here, we report the cryo-electron microscopy structure of the active, C-terminal cleavage product of human DELE1 at ∼3.8 Å resolution, revealing that DELE1 assembles into a high-order oligomer. We confirmed that this C-terminal fragment of DELE1 also assembles into large oligomers in cells. Structurally, the oligomer consists of eight DELE1 monomers that assemble with D4 symmetry via two sets of distinct hydrophobic inter-subunit interactions. We identified the key residues involved in DELE1 oligomerization and confirmed their role in stabilizing the octamer in vitro and in cells using mutagenesis. Further, we show that assembly-impaired DELE1 mutants are compromised in their ability to induce ISR activation in cell culture models. Together, our findings provide molecular insights into the activity of DELE1 and how it signals to promote ISR activity following mitochondrial insult.

Regulation of Intersubunit Interactions in Homotetramer of Glyceraldehyde-3-Phosphate Dehydrogenases upon Its Immobilization in Protein-Kappa-Carrageenan Gels.

Polysaccharides, being biocompatible and biodegradable polymers, are highly attractive as materials for protein delivery systems. However, protein-polysaccharide interactions may lead to protein structural transformation. In the current study, we analyze the structural adjustment of a homotetrameric protein, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), upon its interactions with both flexible coil chain and the rigid helix of κ-carrageenan. FTIR spectroscopy was used to probe the secondary structures of both protein and polysaccharide. Electrostatically driven protein-polysaccharide interactions in dilute solutions resulted in an insoluble complex formation with a constant κ-carrageenan/GAPDH ratio of 0.2, which amounts to 75 disaccharide units per mole of protein tetramer. Upon interactions with both coiled and helical polysaccharides, a weakening of the intersubunit interactions was revealed and attributed to a partial GAPDH tetramer dissociation. In turn, protein distorted the helical conformation of κ-carrageenan when co-gelled. Molecular modeling showed the energy favorable interactions between κ-carrageenan and GAPDH at different levels of oligomerization. κ-Carrageenan binds in the region of the NAD-binding groove and the S-loop in OR contact, which may stabilize the OP dimers. The obtained results highlight the mutual conformational adjustment of oligomeric GAPDH and κ-carrageenan upon interaction and the stabilization of GAPDH's dissociated forms upon immobilization in polysaccharide gels.

Extracellular intersubunit interactions modulate epithelial Na+ channel gating

Epithelial Na+ channels (ENaCs) and related channels have large extracellular domains where specific factors interact and induce conformational changes, leading to altered channel activity. However, extracellular structural transitions associated with changes in ENaC activity are not well defined. Using crosslinking and two-electrode voltage clamp in Xenopus oocytes, we identified several pairs of functional intersubunit contacts where mouse ENaC activity was modulated by inducing or breaking a disulfide bond between introduced Cys residues. Specifically, crosslinking E499C in the β-subunit palm domain and N510C in the α-subunit palm domain activated ENaC, whereas crosslinking βE499C with αQ441C in the α-subunit thumb domain inhibited ENaC. We determined that bridging βE499C to αN510C or αQ441C altered the Na+ self-inhibition response via distinct mechanisms. Similar to bridging βE499C and αQ441C, we found that crosslinking palm domain αE557C with thumb domain γQ398C strongly inhibited ENaC activity. In conclusion, we propose that certain residues at specific subunit interfaces form microswitches that convey a conformational wave during ENaC gating and its regulation.

Assembly and architecture of the type III secretion sorting platform

Type III secretion systems are bacterial nanomachines specialized in protein delivery into target eukaryotic cells. The structural and functional complexity of these machines demands highly coordinated mechanisms for their assembly and operation. The sorting platform is a critical component of type III secretion machines that ensures the timely engagement and secretion of proteins destined to travel this export pathway. However, the mechanisms that lead to the assembly of this multicomponent structure have not been elucidated. Herein, employing an extensive in vivo cross-linking strategy aided by structure modeling, we provide a detailed intersubunit contact survey of the entire sorting platform complex. Using the identified cross-links as signatures for pairwise intersubunit interactions in combination with systematic genetic deletions, we mapped the assembly process of this unique bacterial structure. Insights generated by this study could serve as the bases for the rational development of antivirulence strategies to combat several medically important bacterial pathogens.

Defective ryanodine receptor N-terminus inter-subunit interaction is a common mechanism in neuromuscular and cardiac disorders.

The ryanodine receptor (RyR) is a homotetrameric channel mediating sarcoplasmic reticulum Ca2+ release required for skeletal and cardiac muscle contraction. Mutations in RyR1 and RyR2 lead to life-threatening malignant hyperthermia episodes and ventricular tachycardia, respectively. In this brief report, we use chemical cross-linking to demonstrate that pathogenic RyR1 R163C and RyR2 R169Q mutations reduce N-terminus domain (NTD) tetramerization. Introduction of positively-charged residues (Q168R, M399R) in the NTD-NTD inter-subunit interface normalizes RyR2-R169Q NTD tetramerization. These results indicate that perturbation of NTD-NTD inter-subunit interactions is an underlying molecular mechanism in both RyR1 and RyR2 pathophysiology. Importantly, our data provide proof of concept that stabilization of this critical RyR1/2 structure-function parameter offers clear therapeutic potential.

Ligand-mediated Structural Dynamics of a Mammalian Pancreatic KATP Channel

Regulation of pancreatic KATP channels involves orchestrated interactions of their subunits, Kir6.2 and SUR1, and ligands. Previously we reported KATP channel cryo-EM structures in the presence and absence of pharmacological inhibitors and ATP, focusing on the mechanisms by which inhibitors act as pharmacological chaperones of KATP channels (Martin et al., 2019). Here we analyzed the same cryo-EM datasets with a focus on channel conformational dynamics to elucidate structural correlates pertinent to ligand interactions and channel gating. We found pharmacological inhibitors and ATP enrich a channel conformation in which the Kir6.2 cytoplasmic domain is closely associated with the transmembrane domain, while depleting one where the Kir6.2 cytoplasmic domain is extended away into the cytoplasm. This conformational change remodels a network of intra- and inter-subunit interactions as well as the ATP and PIP2 binding pockets. The structures resolved key contacts between the distal N-terminus of Kir6.2 and SUR1′s ABC module involving residues implicated in channel function and showed a SUR1 residue, K134, participates in PIP2 binding. Molecular dynamics simulations revealed two Kir6.2 residues, K39 and R54, that mediate both ATP and PIP2 binding, suggesting a mechanism for competitive gating by ATP and PIP2.

Molecular basis for the regulation of human glycogen synthase by phosphorylation and glucose-6-phosphate

Glycogen synthase (GYS1) is the central enzyme in muscle glycogen biosynthesis. GYS1 activity is inhibited by phosphorylation of its amino (N) and carboxyl (C) termini, which is relieved by allosteric activation of glucose-6-phosphate (Glc6P). We present cryo-EM structures at 3.0–4.0 Å resolution of phosphorylated human GYS1, in complex with a minimal interacting region of glycogenin, in the inhibited, activated and catalytically competent states. Phosphorylations of specific terminal residues are sensed by different arginine clusters, locking the GYS1 tetramer in an inhibited state via intersubunit interactions. The Glc6P activator promotes conformational change by disrupting these interactions and increases the flexibility of GYS1, such that it is poised to adopt a catalytically competent state when the sugar donor UDP-glucose (UDP-glc) binds. We also identify an inhibited-like conformation that has not transitioned into the activated state, in which the locking interaction of phosphorylation with the arginine cluster impedes subsequent conformational changes due to Glc6P binding. Our results address longstanding questions regarding the mechanism of human GYS1 regulation.

Structural Enzymology of the Cysteine Tryptophylquinone (CTQ) Enzyme GoxA

GoxA from Pseudoalteromonas luteoviolaceae belongs to a recently identified family of enzymes (the LodA‐like proteins) that utilize a post‐translationally derived cysteine tryptophylquinone (CTQ) cofactor for catalysis. In contrast to other tryptophylquinone enzymes, GoxA acts as an oxidase rather than a dehydrogenase, using molecular oxygen to catalyze the oxidative deamination of glycine to produce ammonia, glyoxylate and hydrogen peroxide. The crystal structure of this enzyme revealed a homotetramer with a novel alpha helical domain and extensive inter‐subunit interactions, which account for the positive cooperativity observed toward its substrate by steady‐state kinetics. Soaking GoxA crystals in glycine results in a dramatic color change from yellow to blue, indicating the formation of an unusual intermediate at the CTQ site that was also detected in solution. The structure of this intermediate reveals a Schiff‐base formed between the substrate and CTQ that is stabilized by numerous hydrogen bond contacts with residues both in the active site and from a neighboring protomer. The kinetics and structures of active site mutants reveal the roles of specific residues in substrate binding, cooperativity, and catalysis, and allowed for the detection of another intermediate in the GoxA reaction pathway. This combination of kinetics, spectroscopy and X‐ray crystallography has allowed for the construction of a detailed reaction mechanism for this unusual enzyme with high‐resolution structures now available for key intermediates.

Intersubunit and intrasubunit interactions driving the MukBEF ATPase

MukBEF, a structural maintenance of chromosome-like protein complex consisting of an ATPase, MukB, and two interacting subunits, MukE and MukF, functions as the bacterial condensin. It is likely that MukBEF compacts DNA via an ATP hydrolysis–dependent DNA loop–extrusion reaction similar to that demonstrated for the yeast structural maintenance of chromosome proteins condensin and cohesin. MukB also interacts with the ParC subunit of the cellular chromosomal decatenase topoisomerase IV, an interaction that is required for proper chromosome condensation and segregation in Escherichia coli, although it suppresses the MukB ATPase activity. Other structural determinants and interactions that regulate the ATPase activity of MukBEF are not clear. Here, we have investigated the MukBEF ATPase activity, identifying intersubunit and intrasubunit interactions by protein–protein crosslinking and site-specific mutagenesis. We show that interactions between the hinge of MukB and its neck region are essential for the ATPase activity, that the ParC subunit of topoisomerase IV inhibits the MukB ATPase by preventing this interaction, that MukE interaction with DNA is likely essential for viability, and that interactions between MukF and the MukB neck region are necessary for ATPase activity and viability.

The phosphorylation and dephosphorylation switch of VCP/p97 regulates the architecture of centrosome and spindle.

The proper orientation of centrosome and spindle is essential for genome stability; however, the mechanism that governs these processes remains elusive. Here, we demonstrated that polo-like kinase 1 (Plk1), a key mitotic kinase, phosphorylates residue Thr76 in VCP/p97 (an AAA-ATPase), at the centrosome from prophase to anaphase. This phosphorylation process recruits VCP to the centrosome and in this way, it regulates centrosome orientation. VCP exhibits strong co-localization with Eg5 (a mitotic kinesin motor), at the mitotic spindle, and the dephosphorylation of Thr76 in VCP is required for the enrichment of both VCP and Eg5 at the spindle, thus ensuring proper spindle architecture and chromosome segregation. We also showed that the phosphatase, PTEN, is responsible for the dephosphorylation of Thr76 in VCP; when PTEN was knocked down, the normal spread of VCP from the centrosome to the spindle was abolished. Cryo-EM structures of VCPT76A and VCPT76E, which represent dephosphorylated and phosphorylated states of VCP, respectively, revealed that the Thr76 phosphorylation modulates VCP by altering the inter-domain and inter-subunit interactions, and ultimately the nucleotide-binding pocket conformation. Interestingly, the tumor growth in nude mice implanted with VCPT76A-reconstituted cancer cells was significantly slower when compared with those implanted with VCPWT-reconstituted cancer cells. Collectively, our findings demonstrate that the phosphorylation and dephosphorylation switch of VCP regulates the architecture of centrosome and spindle for faithful chromosome segregation.

Relative domain orientation of the L289K HIV-1 reverse transcriptase monomer.

HIV-1 reverse transcriptase (RT) is a heterodimer comprised p66 and p51 subunits (p66/p51). Several single amino acid substitutions in RT, including L289K, decrease p66/p51 dimer affinity, and reduce enzymatic functioning. Here, small-angle X-ray scattering (SAXS) with proton paramagnetic relaxation enhancement (PRE), 19 F site-specific NMR, and size exclusion chromatography (SEC) were performed for the p66 monomer with the L289K mutation, p66L289K . NMR and SAXS experiments clearly elucidated that the thumb and RNH domains in the monomer do not rigidly interact with each other but are spatially close to the RNH domain. Based on this structural model of the monomer, p66L289K and p51 were predicted to form a heterodimer while p66 and p51L289K not. We tested this hypothesis by SEC analysis of p66 and p51 containing L289K in different combinations and clearly demonstrated that L289K substitution in the p51 subunit, but not in the p66 subunit, reduces p66/p51 formation. Based on the derived monomer model and the importance of the inter-subunit RNH-thumb domain interaction in p66/p51, validated by SEC, the mechanism of p66 homodimer formation was discussed.

Structural origin of cooperativity in human hemoglobin: a view from different roles of α and β subunits in the α2β2 tetramer.

This mini-review, mainly based on our resonance Raman studies on the structural origin of cooperative O2 binding in human adult hemoglobin (HbA), aims to answering why HbA is a tetramer consisting of two α and two β subunits. Here, we focus on the Fe-His bond, the sole coordination bond connecting heme to a globin. The Fe-His stretching frequencies reflect the O2 affinity and also the magnitude of strain imposed through globin by inter-subunit interactions, which is the origin of cooperativity. Cooperativity was first explained by Monod, Wyman, and Changeux, referred to as the MWC theory, but later explained by the two tertiary states (TTS) theory. Here, we related the higher-order structures of globin observed mainly by vibrational spectroscopy to the MWC theory. It became clear from the recent spectroscopic studies, X-ray crystallographic analysis, and mutagenesis experiments that the Fe-His bonds exhibit different roles between the α and β subunits. The absence of the Fe-His bond in the α subunit in some mutant and artificial Hbs inhibits T to R quaternary structural change upon O2 binding. However, its absence from the β subunit in mutant and artificial Hbs simply enhances the O2 affinity of the α subunit. Accordingly, the inter-subunit interactions between α and β subunits are nonsymmetric but substantial for HbA to perform cooperative O2 binding.

Structure-based molecular characterization of the YetL transcription factor from Bacillus subtilis

Bacillus subtilis is a gram-positive bacterium that has developed to coordinate gene expression and to survive against changes of nutrients and toxic chemicals. Flavonoids are exuded by plant cells and are abundant in the soil. To counteract the antibacterial effects of flavonoids, B. subtilis expresses flavonoid-detoxifying enzymes, and their expression is negatively regulated by transcription factors, including YetL. YetL was shown to control B. subtilis growth through the promoter regions of yetL and yetM genes in response to some flavonoids. Despite the functional significance of the YetL transcription factor in bacterial survival, no structural information is available for YetL. Here, we report the crystal structure of YetL and propose a flavonoid-induced regulatory mechanism. The YetL structure contains the canonical winged helix-turn-helix motif of the MarR superfamily but distinctly presents an additional N-terminal helix. In the dimeric assembly of YetL, the H1 helix intersects the YetL dimer and contributes to extensive intersubunit interactions. As a transcription factor, YetL recognizes a 28-mer operator of double-stranded DNA that contains a palindromic sequence. Moreover, our comparative structural analysis of YetL and other MarR members allows us to propose a flavonoid-induced transcription regulatory mechanism that is used for bacterial adaptation to environmental changes and stresses.

Reconstitution of the DTX3L-PARP9 complex reveals determinants for high-affinity heterodimerization and multimeric assembly.

Ubiquitination and ADP-ribosylation are post-translational modifications that play major roles in pathways including the DNA damage response and viral infection. The enzymes responsible for these modifications are therefore potential targets for therapeutic intervention. DTX3L is an E3 Ubiquitin ligase that forms a heterodimer with PARP9. In addition to its ubiquitin ligase activity, DTX3L-PARP9 also acts as an ADP-ribosyl transferase for Gly76 on the C-terminus of ubiquitin. NAD+-dependent ADP-ribosylation of ubiquitin by DTX3L-PARP9 prevents ubiquitin from conjugating to protein substrates. To gain insight into how DTX3L-PARP9 generates these post-translational modifications, we produced recombinant forms of DTX3L and PARP9 and studied their physical interactions. We show the DTX3L D3 domain (230-510) mediates the interaction with PARP9 with nanomolar affinity and an apparent 1 : 1 stoichiometry. We also show that DTX3L and PARP9 assemble into a higher molecular weight oligomer, and that this is mediated by the DTX3L N-terminal region (1-200). Lastly, we show that ADP-ribosylation of ubiquitin at Gly76 is reversible in vitro by several Macrodomain-type hydrolases. Our study provides a framework to understand how DTX3L-PARP9 mediates ADP-ribosylation and ubiquitination through both intra- and inter-subunit interactions.