Hinge Loop Research Articles

Developing New Peptides and Peptide-Drug Conjugates for Targeting the FGFR2 Receptor-Expressing Tumor Cells and 3D Spheroids.

In this work, we utilized a biomimetic approach for targeting KATO (III) tumor cells and 3D tumoroids. Specifically, the binding interactions of the bioactive short peptide sequences ACSAG (A-pep) and LPHVLTPEAGAT (L-pep) with the fibroblast growth factor receptor (FGFR2) kinase domain was investigated for the first time. Both peptides have been shown to be derived from natural resources previously. We then created a new fusion trimer peptide ACSAG-LPHVLTPEAGAT-GASCA (Trimer-pep) and investigated its binding interactions with the FGFR2 kinase domain in order to target the fibroblast growth factor receptor 2 (FGFR2), which is many overexpressed in tumor cells. Molecular docking and molecular dynamics simulation studies revealed critical interactions with the activation loop, hinge and glycine-rich loop regions of the FGFR2 kinase domain. To develop these peptides for drug delivery, DOX (Doxorubicin) conjugates of the peptides were created. Furthermore, the binding of the peptides with the kinase domain was further confirmed through surface plasmon resonance studies. Cell studies with gastric cancer cells (KATO III) revealed that the conjugates and the peptides induced higher cytotoxicity in the tumor cells compared to normal cells. Following confirmation of cytotoxicity against tumor cells, the ability of the conjugates and the peptides to penetrate 3D spheroids was investigated by evaluating their permeation in co-cultured spheroids grown with KATO (III) and colon tumor-associated fibroblasts (CAFs). Results demonstrated that Trimer-pep conjugated with DOX showed the highest permeation, while the ACSAG conjugate also demonstrated reasonable permeation of the drug. These results indicate that these peptides may be further explored and potentially utilized to create drug conjugates for targeting tumor cells expressing FGFR2 for developing therapeutics.

A measles-vectored vaccine candidate expressing prefusion-stabilized SARS-CoV-2 spike protein brought to phase I/II clinical trials: candidate selection in a preclinical murine model.

In the early COVID-19 pandemic with urgent need for countermeasures, we aimed at developing a replicating viral vaccine using the highly efficacious measles vaccine as vector, a promising technology with prior clinical proof of concept. Building on our successful pre-clinical development of a measles virus (MV)-based vaccine candidate against the related SARS-CoV, we evaluated several recombinant MV expressing codon-optimized SARS-CoV-2 spike glycoprotein. Candidate V591 expressing a prefusion-stabilized spike through introduction of two proline residues in HR1 hinge loop, together with deleted S1/S2 furin cleavage site and additional inactivation of the endoplasmic reticulum retrieval signal, was the most potent in eliciting neutralizing antibodies in mice. After single immunization, V591 induced similar neutralization titers as observed in sera of convalescent patients. The cellular immune response was confirmed to be Th1 skewed. V591 conferred long-lasting protection against SARS-CoV-2 challenge in a murine model with marked decrease in viral RNA load, absence of detectable infectious virus loads, and reduced lesions in the lungs. V591 was furthermore efficacious in an established non-human primate model of disease (see companion article [S. Nambulli, N. Escriou, L. J. Rennick, M. J. Demers, N. L. Tilston-Lunel et al., J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23]). Thus, V591 was taken forward into phase I/II clinical trials in August 2020. Unexpected low immunogenicity in humans (O. Launay, C. Artaud, M. Lachâtre, M. Ait-Ahmed, J. Klein et al., eBioMedicine 75:103810, 2022, https://doi.org/10.1016/j.ebiom.2021.103810) revealed that the underlying mechanisms for resistance or sensitivity to pre-existing anti-measles immunity are not yet understood. Different hypotheses are discussed here, which will be important to investigate for further development of the measles-vectored vaccine platform.IMPORTANCESARS-CoV-2 emerged at the end of 2019 and rapidly spread worldwide causing the COVID-19 pandemic that urgently called for vaccines. We developed a vaccine candidate using the highly efficacious measles vaccine as vector, a technology which has proved highly promising in clinical trials for other pathogens. We report here and in the companion article by Nambulli et al. (J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23) the design, selection, and preclinical efficacy of the V591 vaccine candidate that was moved into clinical development in August 2020, 7 months after the identification of SARS-CoV-2 in Wuhan. These unique in-human trials of a measles vector-based COVID-19 vaccine revealed insufficient immunogenicity, which may be the consequence of previous exposure to the pediatric measles vaccine. The three studies together in mice, primates, and humans provide a unique insight into the measles-vectored vaccine platform, raising potential limitations of surrogate preclinical models and calling for further refinement of the platform.

Structural Dynamics of E6AP E3 Ligase HECT Domain and Involvement of a Flexible Hinge Loop in the Ubiquitin Chain Synthesis Mechanism.

Ubiquitin (Ub) ligases E3 are important factors in selecting target proteins for ubiquitination and determining the type of polyubiquitin chains on the target proteins. In the HECT (homologous to E6AP C-terminus)-type E3 ligases, the HECT domain is composed of an N-lobe and a C-lobe that are connected by a flexible hinge loop. The large conformational rearrangement of the HECT domain via the flexible hinge loop is essential for the HECT-type E3-mediated Ub transfer from E2 to a target protein. However, detailed insights into the structural dynamics of the HECT domain remain unclear. Here, we provide the first direct demonstration of the structural dynamics of the HECT domain using high-speed atomic force microscopy at the nanoscale. We also found that the flexibility of the hinge loop has a great impact not only on its structural dynamics but also on the formation mechanism of free Ub chains.

Identification of residues critical for the extension of Munc18-1 domain 3a

BackgroundNeurotransmitter release depends on the fusion of synaptic vesicles with the presynaptic membrane and is mainly mediated by SNARE complex assembly. During the transition of Munc18-1/Syntaxin-1 to the SNARE complex, the opening of the Syntaxin-1 linker region catalyzed by Munc13-1 leads to the extension of the domain 3a hinge loop, which enables domain 3a to bind SNARE motifs in Synaptobrevin-2 and Syntaxin-1 and template the SNARE complex assembly. However, the exact mechanism of domain 3a extension remains elusive.ResultsHere, we characterized residues on the domain 3a hinge loop that are crucial for the extension of domain 3a by using biophysical and biochemical approaches and electrophysiological recordings. We showed that the mutation of residues T323/M324/R325 disrupted Munc13-1-mediated SNARE complex assembly and membrane fusion starting from Munc18-1/Syntaxin-1 in vitro and caused severe defects in the synaptic exocytosis of mouse cortex neurons in vivo. Moreover, the mutation had no effect on the binding of Synaptobrevin-2 to isolated Munc18-1 or the conformational change of the Syntaxin-1 linker region catalyzed by the Munc13-1 MUN domain. However, the extension of the domain 3a hinge loop in Munc18-1/Syntaxin-1 was completely disrupted by the mutation, leading to the failure of Synaptobrevin-2 binding to Munc18-1/Syntaxin-1.ConclusionsTogether with previous results, our data further support the model that the template function of Munc18-1 in SNARE complex assembly requires the extension of domain 3a, and particular residues in the domain 3a hinge loop are crucial for the autoinhibitory release of domain 3a after the MUN domain opens the Syntaxin-1 linker region.

Monomeric crystal structure of the vaccine carrier protein CRM197 and implications for vaccine development.

CRM197 is a genetically detoxified mutant of diphtheria toxin (DT) that is widely used as a carrier protein in conjugate vaccines. Protective immune responses to several bacterial diseases are obtained by coupling CRM197 to glycans from these pathogens. Wild-type DT has been described in two oligomeric forms: a monomer and a domain-swapped dimer. Their proportions depend on the chemical conditions and especially the pH, with a large kinetic barrier to interconversion. A similar situation occurs in CRM197, where the monomer is preferred for vaccine synthesis. Despite 30 years of research and the increasing application of CRM197 in conjugate vaccines, until now all of its available crystal structures have been dimeric. Here, CRM197 was expressed as a soluble, intracellular protein in an Escherichia coli strain engineered to have an oxidative cytoplasm. The purified product, called EcoCRM, remained monomeric throughout crystallization. The structure of monomeric EcoCRM is reported at 2.0 Å resolution with the domain-swapping hinge loop (residues 379-387) in an extended, exposed conformation, similar to monomeric wild-type DT. The structure enables comparisons across expression systems and across oligomeric states, with implications for monomer-dimer interconversion and for the optimization of conjugation.

Human DND1-RRM2 forms a non-canonical domain-swapped dimer.

RNA Recognition Motifs (RRMs) being the most abundant RNA binding domain in eukaryotes, is a major player in cellular regulation. Human DND1 (dead end protein homolog1) also known as microRNA-mediated repression inhibitor 1 is an RNA binding protein containing two RRMs in tandem. It is essential for migration and viability of primordial germ cells during development, its complete loss causes lethality, and mutations in the gene are associated with perturbed cellular regulation and eventually cancer. Using x-ray crystallography we have determined 2.3 Å structure of the human DND1 RRM2 domain. The structure revealed an interesting non-canonical RRM fold. This is maintained by the formation of a 3D domain-swapped dimer between β1 and β4 strands across protomers. We have delineated the structural basis of the stable domain-swapped dimer formation using the residue level dynamics of protein explored by NMR spectroscopy and MD simulations. Our results indicate that monomer to dimer switch is governed by several determinants such as hinge loop residues, its length, hydrophobicity, concentration of protein, disulphide bond, and so forth. Several variations in the canonical βαββαβ topology have been observed so far. However, this is the first report of domain swapping in the RRM domain, which may increase the binding surface area and allow this protein to exhibit multifaceted role during post-transcriptional regulation. Our structural and dynamics studies substantiate major determinants and molecular basis for domain-swapped dimerization observed in the RRM domain that could allow varied roles for its RRMs.

Molecular Docking, Molecular Dynamics Simulations, and Free Energy Calculation Insights into the Binding Mechanism between VS-4718 and Focal Adhesion Kinase.

Focal adhesion kinase (FAK) is a 125 kDa nonreceptor tyrosine kinase that plays an important role in many carcinomas. Thus, the targeting of FAK by small molecules is considered to be promising for cancer therapy. Some FAK inhibitors have been reported as potential anticancer drugs and have entered into clinical development; for example, VS-4718 is currently undergoing clinical trials. However, the lack of crystal structural data for the binding of VS-4718 with FAK has hindered the optimization of this anticancer agent. In this work, the VS-4718/FAK interaction model was obtained by molecular docking and molecular dynamics simulations. The binding free energies of VS-4718/FAK were also calculated using the molecular mechanics generalized Born surface area method. It was found that the aminopyrimidine group formed hydrogen bonds with the C502 residue of the hinge loop, while the D564 residue of the T-loop interacted with the amide group. In addition, I428, A452, V484, M499, G505, and L553 residues formed hydrophobic interactions with VS-4718. The obtained results therefore provide an improved understanding of the interaction between human FAK and VS-4718. Based on the obtained binding mechanism, 47 novel compounds were designed to target the adenosine 5′-triphosphate-binding pocket of human FAK, and ensemble docking was performed to assess the effects of these modifications on the inhibitor binding affinity. This work is also expected to provide additional insights into potential future target design strategies based on VS-4718.

Dimerization Tendency of 3CLpros of Human Coronaviruses Based on the X-ray Crystal Structure of the Catalytic Domain of SARS-CoV-2 3CLpro.

3CLpro of SARS-CoV-2 is a promising target for developing anti-COVID19 agents. In order to evaluate the catalytic activity of 3CLpros according to the presence or absence of the dimerization domain, two forms had been purified and tested. Enzyme kinetic studies with a FRET method revealed that the catalytic domain alone presents enzymatic activity, despite it being approximately 8.6 times less than that in the full domain. The catalytic domain was crystallized and its X-ray crystal structure has been determined to 2.3 Å resolution. There are four protomers in the asymmetric unit. Intriguingly, they were packed as a dimer though the dimerization domain was absent. The RMSD of superimposed two catalytic domains was 0.190 for 182 Cα atoms. A part of the long hinge loop (LH-loop) from Gln189 to Asp197 was not built in the model due to its flexibility. The crystal structure indicates that the decreased proteolytic activity of the catalytic domain was due to the incomplete construction of the substrate binding part built by the LH-loop. A structural survey with other 3CLpros showed that SARS-CoV families do not have interactions between DM-loop due to the conformational difference at the last turn of helix α7 compared with others. Therefore, we can conclude that the monomeric form contains nascent enzyme activity and that its efficiency increases by dimerization. This new insight may contribute to understanding the behavior of SARS-CoV-2 3CLpro and thus be useful in developing anti-COVID-19 agents.

Hinge region mediates signal transmission of luteinizing hormone and chorionic gonadotropin receptor

Luteinizing hormone-choriogonadotropin receptor (LHCGR), a class A G protein-coupled receptor (GPCR), plays a pivotal role in the maturation of reproductive organs and embryonic development. Compared with other GPCRs, the subfamily of LHCGR has a large extracellular domain (ECD) to interact with glycoprotein hormones. A unique hinge region connects the ECD and transmembrane domain (TMD) to transfer the activation signal. However, the signal transmission mechanism remains largely unknown. Here, both molecular dynamics simulation and evolutional analysis were applied to explore the effect of the hinge region on signal transmission. The glycoprotein hormone determined specific hinge region conformations, including the position of a long hinge loop and the ECD-TMD interface. With the hormone, the hinge region showed a characteristic rotation and displayed an active-like conformational landscape of the ECD-TMD interface with an extended TMD. The active-like hinge region conformation transduces the hormone binding signal downwards from ECD to TMD. The relationship between the hinge region and the intracelluar G protein-binding pocket was also inferred. The hinge region-mediated signal transmission mechanism offers a deeper understanding of LHCGR and provides insights into the elucidation of GPCR activation.

Structures of full-length glycoprotein hormone receptor signalling complexes.

Luteinizing hormone and chorionic gonadotropin are glycoprotein hormones that are related to follicle-stimulating hormone and thyroid-stimulating hormone1,2. Luteinizing hormone and chorionic gonadotropin are essential to human reproduction and are important therapeutic drugs3-6. They activate the same G-protein-coupled receptor, luteinizing hormone-choriogonadotropin receptor (LHCGR), by binding to the large extracellular domain3. Here we report four cryo-electron microscopy structures of LHCGR: two structures of the wild-type receptor in the inactive and active states; and two structures of the constitutively active mutated receptor. The active structures are bound to chorionic gonadotropin and the stimulatory G protein (Gs), and one of the structures also contains Org43553, an allosteric agonist7. The structures reveal a distinct 'push-and-pull' mechanism of receptor activation, in which the extracellular domain is pushed by the bound hormone and pulled by the extended hinge loop next to the transmembrane domain. A highly conserved 10-residue fragment (P10) from the hinge C-terminal loop at the interface between the extracellular domain and the transmembrane domain functions as a tethered agonist to induce conformational changes in the transmembrane domain and G-protein coupling. Org43553 binds to a pocket of the transmembrane domain and interacts directly with P10, which further stabilizes the active conformation. Together, these structures provide a common model for understanding the signalling of glycoprotein hormone receptors and a basis for drug discovery for endocrine diseases.

The Molecular Mechanism of Domain Swapping of the C-Terminal Domain of the SARS-Coronavirus Main Protease

In three-dimensional domain swapping, two protein monomers exchange a part of their structures to form an intertwined homodimer, whose subunits resemble the monomer. Several viral proteins domain swap to increase their structural complexity or functional avidity. The main protease (Mpro) of the severe acute respiratory syndrome (SARS) coronavirus proteolyzes viral polyproteins and has been a target for anti-SARS drug design. Domain swapping in the α-helical C-terminal domain of Mpro (MproC) locks Mpro into a hyperactive octameric form that is hypothesized to promote the early stages of viral replication. However, in the absence of a complete molecular understanding of the mechanism of domain swapping, investigations into the biological relevance of this octameric Mpro have stalled. Isolated MproC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of MproC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of MproC folding. Further, we find that a contact between a tryptophan in the MproC domain-swapping hinge and an arginine elsewhere forms early during folding, modulates the folding route, and promotes domain swapping to the native structure. An examination of the sequence and the structure of the tryptophan containing hinge loop shows that it has a propensity to form multiple secondary structures and contacts, indicating that it could be stabilized into either the monomer- or dimer-promoting conformations by mutations or ligand binding. Finally, because all residues in the tryptophan loop are identical in SARS-CoV and SARS-CoV-2, mutations that modulate domain swapping may provide insights into the role of octameric Mpro in the early-stage viral replication of both viruses.

Thermodynamic Control of Domain Swapping by Modulating the Helical Propensity in the Hinge Region of Myoglobin.

Domain swapping is an exception to Anfinsen's dogma, and more than one structure can be produced from the same amino acid sequence by domain swapping. We have previously shown that myoglobin (Mb) can form a domain-swapped dimer in which the hinge region is converted to a helical structure. In this study, we showed that domain-swapped dimerization of Mb was achieved by a single Ala mutation of Gly at position 80. Multiple Ala mutations at positions 81 and 82 in addition to position 80 facilitated dimerization of Mb by stabilization of the dimeric states. Domain swapping tendencies correlated well with the helical propensity of the mutated residue in a series of Mb mutants with amino acids introduced to the hinge region. These findings demonstrate that a single mutation in the hinge loop to modify helical propensity can control oligomer formation, providing new ideas to create high-order protein oligomers using domain swapping.

Structure-Based Design of Prefusion-Stabilized Filovirus Glycoprotein Trimers

Ebola virus causes severe hemorrhagic fever, often leading to death in humans. The trimeric fusion glycoprotein (GP) is the sole target for neutralizing antibodies and is the major focus of vaccine development. Soluble GP ectodomains are unstable and mostly monomeric when not fused to a heterologous trimerization domain. Here, we report structure-based designs of Ebola and Marburg GP trimers based on a stabilizing mutation in the hinge loop in refolding region 1 and substitution of a partially buried charge at the interface of the GP1 and GP2 subunits. The combined substitutions (T577P and K588F) substantially increased trimer expression for Ebola GP proteins. We determined the crystal structure of stabilized GP from the Makona Zaire ebolavirus strain without a trimerization domain or complexed ligand. The structure reveals that the stabilized GP adopts the same trimeric prefusion conformation, provides insight into triggering of GP conformational changes, and should inform future filovirus vaccine development.

Comparing Structure and Conformation of Diphtheria Toxin With Its Non-toxic Mutant (E349K) at 300 K Using Molecular Dynamics Simulations

Background: The molecular dynamics simulations have provided detailed information on the fluctuations and conformational changes of proteins. Mutation of GLu349 of diphtheria toxin (DT) to Lys inhibits the molecular cytotoxicity in mammalian cells. In this work, we thus aimed to evaluate the effects of the mutation on the structure and the conformation of DT using molecular dynamic simulations. Methods: PYMOL system was used for introducing the mutant amino acid to DT. Three dimensional structures were visualized using YASARA. The protonation fixing process was done using H++ server. SPD viewer was then applied to retrieve missing hydrogen atoms. Molecular dynamic simulations were also carried out using GROMACS software. Finally, RMSF, Rg, and RMSD graphs were drawn by Sigma Plot using GROMACS outputs. Results: The results of our analysis indicated that amino acid residue fluctuations in the catalytic domain (C) of DT were greater than those of its mutant (E349K). Moreover, residue fluctuations in the region, including Ile344- val347, Tyr514-Ser525, and Ile533-Lys534 of DT were greater than those of the mutant (E349K). However, residue fluctuations in the region including Cys186-Cys201 and Glu349-Val351 of DT were lower than those of E349K. In addition, the disulfide bridge (Cys186–Cys201) was formed in DT, whereas it was not observed in the mutant. The secondary structure analysis showed that the beta-sheet content of E349K decreased compared with DT. Additionally, the conformation of DT was different from that of E349K in the hinge loop regions (Ala379–Thr386 and Tyr514-Ser525). The radius of gyration (Rg) and the root mean square deviation (RMSD) of E349K were greater than those of DT. The conformational stability and compactness of DT were higher than those of E349K. Conclusions: The method used in this study can demonstrate the structural details of toxins as important aspects to predict activities of the receptor binding and the catalytic domains. Therefore, it can be used to evaluate conformational changes of toxins as well as other vaccine candidates.

Crystal structure of the Fab region of a neutralizing antibody against granulocyte-macrophage colony-stimulating factor.

An increased level of granulocyte-macrophage colony-stimulating factor has a potential role in the development of autoimmune diseases, and the neutralization of its activity by monoclonal antibodies is a promising therapy for some diseases. Here, the crystal structure of the Fab region of EV1007, a fully human antibody expressed in Chinese hamster ovary cells that was developed from human peripheral blood mononuclear cells, is described. The structure closely resembles that of MB007, which is the Fab region of the same antibody expressed in Escherichia coli [Blech et al. (2012), Biochem. J. 447, 205-215], except at the hinge regions between the immunoglobulin domains and the H3 loop region. This paper presents evidence for the flexibility of the hinge and H3 loop regions of the antibody based on the comparison of two independently solved crystal structures.

Abstract 2758: Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor

Abstract The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers, including hepatocellular carcinoma, pancreatic cancer, oral cancer, breast cancer and non-small cell lung cancer. However, the functional mechanisms of human HDGF had been puzzling because of a lack of essential knowledge of its intact structure in complex with DNA. We present the first crystal structures of the human HDGF PWWP domain (residues 1 - 100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four β-strands and two α-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable α-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions. Note: This abstract was not presented at the meeting. Citation Format: Chun-Jung Chen, Li-Jin Hsu. Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2758.

TADOSS: computational estimation of tandem domain swap stability.

SummaryProteins with highly similar tandem domains have shown an increased propensity for misfolding and aggregation. Several molecular explanations have been put forward, such as swapping of adjacent domains, but there is a lack of computational tools to systematically analyze them. We present the TAndem DOmain Swap Stability predictor (TADOSS), a method to computationally estimate the stability of tandem domain-swapped conformations from the structures of single domains, based on previous coarse-grained simulation studies. The tool is able to discriminate domains susceptible to domain swapping and to identify structural regions with high propensity to form hinge loops. TADOSS is a scalable method and suitable for large scale analyses.Availability and implementationSource code and documentation are freely available under an MIT license on GitHub at https://github.com/lafita/tadoss.Supplementary information Supplementary data are available at Bioinformatics online.

Structural plasticity of the HHD2 domain of whirlin.

Whirlin is a protein essential to sensory neurons. Its defects are responsible for nonsyndromic deafness or for the Usher syndrome, a condition associating congenital deafness and progressive blindness. This large multidomain scaffolding protein is expressed in three isoforms with different functions and localizations in stereocilia bundles of hearing hair cells or in the connecting cilia of photoreceptor cells. The HHD2 domain of whirlin is the only domain shared by all isoforms, but its function remains unknown. In this article, we report its crystal structure in two distinct conformations, a monomeric five-helix bundle, similar to the known structure of other HHD domains, and a three-helix bundle organized as a swapped dimer. Most of the hydrophobic contacts and electrostatic interactions that maintain the globular monomeric form are conserved at the protomer interface of the dimer. NMR experiments revealed that the five-helix conformation is predominant in solution, but exhibits increased dynamics on one face encompassing the hinge loops. Using NMR and SAXS, we also show that HHD2 does not interact with its preceding domains. Our findings suggest that structural plasticity might play a role in the function of the HHD2 domain.

The forkhead domain hinge-loop plays a pivotal role in DNA binding and transcriptional activity of FOXP2.

Forkhead box (FOX) proteins are a ubiquitously expressed family of transcription factors that regulate the development and differentiation of a wide range of tissues in animals. The FOXP subfamily members are the only known FOX proteins capable of forming domain-swapped forkhead domain (FHD) dimers. This is proposed to be due to an evolutionary mutation (P539A) that lies in the FHD hinge loop, a key region thought to fine-tune DNA sequence specificity in the FOX transcription factors. Considering the importance of the hinge loop in both the dimerisation mechanism of the FOXP FHD and its role in tuning DNA binding, a detailed investigation into the implications of mutations within this region could provide important insight into the evolution of the FOX family. Isothermal titration calorimetry and hydrogen exchange mass spectroscopy were used to study the thermodynamic binding signature and changes in backbone dynamics of FOXP2 FHD DNA binding. Dual luciferase reporter assays were performed to study the effect that the hinge-loop mutation has on FOXP2 transcriptional activity in vivo. We demonstrate that the change in dynamics of the hinge-loop region of FOXP2 alters the energetics and mechanism of DNA binding highlighting the critical role of hinge loop mutations in regulating DNA binding characteristics of the FOX proteins.

Structural basis of mitochondrial receptor binding and constriction by DRP1.

SummaryMitochondrial inheritance, genome maintenance, and metabolic adaptation depend on organelle fission by Dynamin-Related Protein 1 (DRP1) and its mitochondrial receptors. DRP1 receptors include the paralogs Mitochondrial Dynamics 49 and 51 (MID49/MID51) and Mitochondrial Fission Factor (MFF), but the mechanisms by which these proteins recruit and regulate DRP1 are unknown. Here we present a cryoEM structure of human, full-length DRP1 coassembled with MID49 and an analysis of structure- and disease-based mutations. We report that GTP induces a remarkable elongation and rotation of the G-domain, Bundle-Signaling Element (BSE) and connecting hinge loops of DRP1. In this conformation, a network of multivalent interactions promotes polymerization of a linear DRP1 filament with MID49/MID51. Following coassembly, GTP hydrolysis and exchange lead to MID receptor dissociation, filament shortening and curling of DRP1 oligomers into constricted and closed rings. Together, these views of full-length, receptor- and nucleotide-bound conformations reveal how DRP1 performs mechanical work through nucleotide-driven allostery.