Showing papers on "Conformational change published in 2017"
TL;DR: This phenomenon could be extended to other betacoronaviruses utilizing CTD1 of the S1 subunit for receptor binding, which provides new insights into the intermediate states of coronavirus pre-fusion spike trimer during infection.
Abstract: The global outbreak of SARS in 2002-2003 was caused by the infection of a new human coronavirus SARS-CoV. The infection of SARS-CoV is mediated mainly through the viral surface glycoproteins, which consist of S1 and S2 subunits and form trimer spikes on the envelope of the virions. Here we report the ectodomain structures of the SARS-CoV surface spike trimer in different conformational states determined by single-particle cryo-electron microscopy. The conformation 1 determined at 4.3 A resolution is three-fold symmetric and has all the three receptor-binding C-terminal domain 1 (CTD1s) of the S1 subunits in "down" positions. The binding of the "down" CTD1s to the SARS-CoV receptor ACE2 is not possible due to steric clashes, suggesting that the conformation 1 represents a receptor-binding inactive state. Conformations 2-4 determined at 7.3, 5.7 and 6.8 A resolutions are all asymmetric, in which one RBD rotates away from the "down" position by different angles to an "up" position. The "up" CTD1 exposes the receptor-binding site for ACE2 engagement, suggesting that the conformations 2-4 represent a receptor-binding active state. This conformational change is also required for the binding of SARS-CoV neutralizing antibodies targeting the CTD1. This phenomenon could be extended to other betacoronaviruses utilizing CTD1 of the S1 subunit for receptor binding, which provides new insights into the intermediate states of coronavirus pre-fusion spike trimer during infection.
537 citations
TL;DR: The finding that T163S mutants of the β subunit confer resistance to Ca2+‐induced, PTP‐dependent death in cells and developing zebrafish embryos is a major advance in the molecular definition of the transition of F‐ATP synthase to a channel and of its role in cell death.
Abstract: F-ATP synthases convert the electrochemical energy of the H+ gradient into the chemical energy of ATP with remarkable efficiency. Mitochondrial F-ATP synthases can also undergo a Ca2+-dependent transformation to form channels with properties matching those of the permeability transition pore (PTP), a key player in cell death. The Ca2+ binding site and the mechanism(s) through which Ca2+ can transform the energy-conserving enzyme into a dissipative structure promoting cell death remain unknown. Through in vitro, in vivo and in silico studies we (i) pinpoint the "Ca2+-trigger site" of the PTP to the catalytic site of the F-ATP synthase β subunit and (ii) define a conformational change that propagates from the catalytic site through OSCP and the lateral stalk to the inner membrane. T163S mutants of the β subunit, which show a selective decrease in Ca2+-ATP hydrolysis, confer resistance to Ca2+-induced, PTP-dependent death in cells and developing zebrafish embryos. These findings are a major advance in the molecular definition of the transition of F-ATP synthase to a channel and of its role in cell death.
148 citations
TL;DR: Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus, West Nile virus, and Yellow fever virus on A549 cells in vivo, with EC50 values in low micromolar range.
Abstract: The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 18 μM, 114 μM, and 48 μM, respectively Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 488 μM on A549 cells Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618
102 citations
TL;DR: A dynamic foldamer scaffold has been ligated to a water-compatible, metal-centred binding site and a conformationally responsive fluorophore to form a receptor mimic that inserts into the membrane of artificial vesicles.
Abstract: A dynamic foldamer scaffold has now been ligated to a water-compatible, metal-centred binding site and a conformationally responsive fluorophore to form a receptor mimic that inserts into the membrane of artificial vesicles. Binding of specific carboxylate ligands induces a global conformational change that depends on the structure of the ligand, and can be detected via fluorescence.
86 citations
TL;DR: Using a combination of biochemical, structural, and cellular approaches, it is demonstrated that the extracellular domains of BTN3A1 adopt a V-shaped conformation at rest, and that locking them in this resting conformation without perturbing their membrane reorganization properties diminish pAg-induced T cell activation.
Abstract: Human Vγ9Vδ2 T cells respond to microbial infections as well as certain types of tumors. The key initiators of Vγ9Vδ2 activation are small, pyrophosphate-containing molecules called phosphoantigens (pAgs) that are present in infected cells or accumulate intracellularly in certain tumor cells. Recent studies demonstrate that initiation of the Vγ9Vδ2 T cell response begins with sensing of pAg via the intracellular domain of the butyrophilin 3A1 (BTN3A1) molecule. However, it is unknown how downstream events can ultimately lead to T cell activation. Here, using NMR spectrometry and molecular dynamics (MD) simulations, we characterize a global conformational change in the B30.2 intracellular domain of BTN3A1 induced by pAg binding. We also reveal by crystallography two distinct dimer interfaces in the BTN3A1 full-length intracellular domain, which are stable in MD simulations. These interfaces lie in close proximity to the pAg-binding pocket and contain clusters of residues that experience major changes of chemical environment upon pAg binding. This suggests that pAg binding disrupts a preexisting conformation of the BTN3A1 intracellular domain. Using a combination of biochemical, structural, and cellular approaches we demonstrate that the extracellular domains of BTN3A1 adopt a V-shaped conformation at rest, and that locking them in this resting conformation without perturbing their membrane reorganization properties diminishes pAg-induced T cell activation. Based on these results, we propose a model in which a conformational change in BTN3A1 is a key event of pAg sensing that ultimately leads to T cell activation.
83 citations
TL;DR: This work constitutes a proof-of-concept study of enzymatic activities using plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.
Abstract: Monitoring the kinetics and conformational dynamics of single enzymes is crucial to better understand their biological functions because these motions and structural dynamics are usually unsynchronized among the molecules. However, detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single-molecule basis remains as a challenge to established optical techniques because of the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually nontrivial, and the labels themselves might skew the physical properties of the enzyme. We demonstrate an optical, label-free method capable of observing enzymatic interactions and associated conformational changes on a single-molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we use two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches, we find that low and high polymerase activities can be clearly discerned through their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities using plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.
75 citations
TL;DR: It is suggested that the conformational change of the syntaxin‐1 linker region induced by Munc13‐1 initiates ternary SNARE complex formation in the neuronal system.
Abstract: The soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor (SNARE) protein syntaxin‐1 adopts a closed conformation when bound to Munc18‐1, preventing binding to synaptobrevin‐2 and SNAP‐25 to form the ternary SNARE complex. Although it is known that the MUN domain of Munc13‐1 catalyzes the transition from the Munc18‐1/syntaxin‐1 complex to the SNARE complex, the molecular mechanism is unclear. Here, we identified two conserved residues (R151, I155) in the syntaxin‐1 linker region as key sites for the MUN domain interaction. This interaction is essential for SNARE complex formation in vitro and synaptic vesicle priming in neuronal cultures. Moreover, this interaction is important for a tripartite Munc18‐1/syntaxin‐1/MUN complex, in which syntaxin‐1 still adopts a closed conformation tightly bound to Munc18‐1, whereas the syntaxin‐1 linker region changes its conformation, similar to that of the LE mutant of syntaxin‐1 when bound to Munc18‐1. We suggest that the conformational change of the syntaxin‐1 linker region induced by Munc13‐1 initiates ternary SNARE complex formation in the neuronal system.
65 citations
TL;DR: The statistical analyses of interaction contacts of the antibiotic ampicillin with residues based on MD trajectories suggest that two Zn ions are essential for maintaining the binding of AMP with NDM-1 and double-Zn coordination exerts strong restriction on the motions of loop L10 relative to loops L3 and L4.
Abstract: The hydrolysis of a β-lactam core ring caused by new Delphi metallo-β-lactamase 1 (NDM-1) with the help of two zinc cofactors induces significant resistance toward β-lactam antibiotics. Molecular dynamics (MD) simulations and the umbrella sampling method are integrated to study the conformational change mechanism of NDM-1 mediated by zinc ion binding. The statistical analyses of interaction contacts of the antibiotic ampicillin (AMP) with residues based on MD trajectories suggest that two Zn ions are essential for maintaining the binding of AMP with NDM-1. Umbrella sampling simulations further reveal that double-Zn coordination exerts strong restriction on the motions of loop L10 relative to loops L3 and L4. Principal component (PC) analysis also demonstrates that zinc ion binding totally inhibits the motion extent of NDM-1 and changes internal motion modes in NDM-1. We expect that the current study can provide significant dynamical information involving conformational changes of NDM-1 for the development of efficient inhibitors to decrease drug resistance of NDM-1 toward antibiotics.
63 citations
TL;DR: Using a biomembrane force probe, the bending and unbending conformational changes of single αVβ3 integrins on living cell surfaces in real-time are characterized, providing insights into how αV β3 acts as a molecular machine and how its physiological function and molecular structure are coupled at the single-molecule level.
60 citations
TL;DR: A molecular description of a high-energy enzyme state in a conformational selection pathway is presented by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography and it is discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state.
Abstract: Proteins can bind target molecules through either induced fit or conformational selection pathways In the conformational selection model, a protein samples a scarcely populated high-energy state that resembles a target-bound conformation In enzymatic catalysis, such high-energy states have been identified as crucial entities for activity and the dynamic interconversion between ground states and high-energy states can constitute the rate-limiting step for catalytic turnover The transient nature of these states has precluded direct observation of their properties Here, we present a molecular description of a high-energy enzyme state in a conformational selection pathway by an experimental strategy centered on NMR spectroscopy, protein engineering, and X-ray crystallography Through the introduction of a disulfide bond, we succeeded in arresting the enzyme adenylate kinase in a closed high-energy conformation that is on-pathway for catalysis A 19-A X-ray structure of the arrested enzyme in complex with a transition state analog shows that catalytic sidechains are properly aligned for catalysis We discovered that the structural sampling of the substrate free enzyme corresponds to the complete amplitude that is associated with formation of the closed and catalytically active state In addition, we found that the trapped high-energy state displayed improved ligand binding affinity, compared with the wild-type enzyme, demonstrating that substrate binding to the high-energy state is not occluded by steric hindrance Finally, we show that quenching of fast time scale motions observed upon ligand binding to adenylate kinase is dominated by enzyme-substrate interactions and not by intramolecular interactions resulting from the conformational change
58 citations
TL;DR: Negative-stain electron microscopy is used to probe conformational states of the entire ectodomain of LRP6 in solution and found that the four-module ectodome undergoes a large bending motion hinged at the junction between the second and the third modules, modulated by evolutionarily conserved N-glycan chains proximal to the joint.
TL;DR: Control over a polypeptide conformation can be achieved by altering the donor-acceptor properties of side-chain triazole units via protonation-deprotonation, as well as designing smart, cell-penetrating polymers that undergo acid-activated endosomal escape in living cells.
Abstract: Synthetic polypeptides have received increasing attention due to their ability to form higher ordered structures similar to proteins. The control over their secondary structures, which enables dynamic conformational changes, is primarily accomplished by tuning the side-chain hydrophobic or ionic interactions. Herein we report a strategy to modulate the conformation of polypeptides utilizing donor–acceptor interactions emanating from side-chain H-bonding ligands. Specifically, 1,2,3-triazole groups, when incorporated onto polypeptide side-chains, serve as both H-bond donors and acceptors at neutral pH and disrupt the α-helical conformation. When protonated, the resulting 1,2,3-triazolium ions lose the ability to act as H-bond acceptors, and the polypeptides regain their α-helical structure. The conformational change of triazole polypeptides in response to the donor-acceptor pattern was conclusively demonstrated using both experimental-based and simulation-based methods. We further showed the utility of this transition by designing smart, cell-penetrating polymers that undergo acid-activated endosomal escape in living cells. Hydrogen bonding plays a major role in determining the tridimensional structure of biopolymers. Here, the authors show that control over a polypeptide conformation can be achieved by altering the donor-acceptor properties of side-chain triazole units via protonation-deprotonation.
TL;DR: 3D structures of a PET model compound bound to the crystal structures were built, and mutated the putative substrate-binding site predicted from the models, and the enzymatic activities of the mutants for the model substrate poly(butylene succinate-co-adipate) were experimentally determined.
TL;DR: Results suggest BTN3A1 selectively detects P-Ag intracellularly via a conformational antigenic sensor in its B30.2 domain and have implications for rational design of antigens for Vγ9/Vδ2-based T-cell immunotherapies.
Abstract: Human Vγ9/Vδ2 T-cells detect tumor cells and microbial infections by recognizing small phosphorylated prenyl metabolites termed phosphoantigens (P–Ag). The type-1 transmembrane protein Butyrophilin 3A1 (BTN3A1) is critical to the P–Ag-mediated activation of Vγ9/Vδ2 T-cells; however, the molecular mechanisms involved in BTN3A1-mediated metabolite sensing are unclear, including how P–Ag’s are discriminated from nonantigenic small molecules. Here, we utilized NMR and X-ray crystallography to probe P–Ag sensing by BTN3A1. Whereas the BTN3A1 immunoglobulin variable domain failed to bind P–Ag, the intracellular B30.2 domain bound a range of negatively charged small molecules, including P–Ag, in a positively charged surface pocket. However, NMR chemical shift perturbations indicated BTN3A1 discriminated P–Ag from nonantigenic small molecules by their ability to induce a specific conformational change in the B30.2 domain that propagated from the P–Ag binding site to distal parts of the domain. These results sugge...
TL;DR: It is demonstrated that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnK allosteric landscape, which is a highly dynamic unit of the structure in all ligand-bound states.
TL;DR: A regulatory mechanism of h TS activity that involves allosteric regulation of interactions of hTS with its own mRNA depending on cellular demands for TMP is proposed.
TL;DR: The latest developments in cryo-EM image analysis methods for conformational variability analysis are the focus of this review.
TL;DR: A combination of NMR measurements and kinetic models based on molecular dynamics simulations are used to rationalize the difference in the membrane permeability of cyclosporine E (CsE) and CsA, finding the most striking difference is found in the interconversion rates between the conformational states favored in water and in chloroform.
Abstract: Cyclic peptides have regained interest as potential inhibitors of challenging targets but have often a low bioavailability. The natural product cyclosporine A (CsA) is the textbook exception. Despite its size and polar backbone, it is able to passively cross membranes. This ability is hypothesized to be due to a conformational change from the low-energy conformation in water to a "congruent" conformation that is populated both in water and inside the membrane. Here, we use a combination of NMR measurements and kinetic models based on molecular dynamics simulations to rationalize the difference in the membrane permeability of cyclosporine E (CsE) and CsA. The structure of CsE differs only in a backbone methylation, but its membrane permeability is one order of magnitude lower. The most striking difference is found in the interconversion rates between the conformational states favored in water and in chloroform, which are up to one order of magnitude slower for CsE compared to CsA.
TL;DR: Results show that ligand binding to BTN3A1 induces a conformational change within the intracellular domain that involves the JM region and is required for full activation of Vγ9Vδ2 T cells.
Abstract: Small isoprenoid diphosphates, such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), are ligands of the internal domain of BTN3A1. Ligand binding in target cells promotes activation of Vγ9...
TL;DR: The data indicate the Fe-NP results in unfolding of Cyt c and subsequent peroxidase-like activity of denatured species is a crucial step for their potential application as intracellular delivery carriers and medicinal agents.
Abstract: Herein, the interaction of iron nanoparticle (Fe-NP) with cytochrome c (Cyt c) was investigated, and a range of techniques such as dynamic light scattering (DLS), zeta potential measurements, static and synchronous fluorescence spectroscopy, near and far circular dichroism (CD) spectroscopy, and ultraviolet–visible (UV–vis) spectroscopy were used to analyze the interaction between Cyt c and Fe-NP. DLS and zeta potential measurements showed that the values of hydrodynamic radius and charge distribution of Fe-NP are 83.95 ± 3.7 nm and 4.5 ± .8 mV, respectively. The fluorescence spectroscopy results demonstrated that the binding of Fe-NP with Cyt c is mediated by hydrogen bonds and van der Waals interactions. Also Fe-NP induced conformational changes in Cyt c and reduced the melting temperature value of Cyt c from 79.18 to 71.33°C. CD experiments of interaction between Fe-NP and Cyt c revealed that the secondary structure of Cyt c with the dominant α-helix structures remained unchanged whereas the tertiary s...
TL;DR: This work adapted hydrogen/deuterium-exchange mass spectrometry to analyze conformational changes in NhaA upon Li+ binding at physiological pH and proposes a model for the ion translocation mechanism that explains previously controversial data for this antiporter.
Abstract: Na+/H+ antiporters comprise a family of membrane proteins evolutionarily conserved in all kingdoms of life and play an essential role in cellular ion homeostasis. The NhaA crystal structure of Escherichia coli has become the paradigm for this class of secondary active transporters. However, structural data are only available at low pH, where NhaA is inactive. Here, we adapted hydrogen/deuterium-exchange mass spectrometry (HDX-MS) to analyze conformational changes in NhaA upon Li+ binding at physiological pH. Our analysis revealed a global conformational change in NhaA with two sets of movements around an immobile binding site. Based on these results, we propose a model for the ion translocation mechanism that explains previously controversial data for this antiporter. Furthermore, these findings contribute to our understanding of related human transporters that have been linked to various diseases.
TL;DR: The authors present the structures of the β-propeller domain at neutral and acidic pH, which reveal that sortilin dimerises and undergoes conformational changes at low pH and further propose a model for low pH-induced ligand release by endocytosis receptors.
Abstract: Low pH-induced ligand release and receptor recycling are important steps for endocytosis. The transmembrane protein sortilin, a β-propeller containing endocytosis receptor, internalizes a diverse set of ligands with roles in cell differentiation and homeostasis. The molecular mechanisms of pH-mediated ligand release and sortilin recycling are unresolved. Here we present crystal structures that show the sortilin luminal segment (s-sortilin) undergoes a conformational change and dimerizes at low pH. The conformational change, within all three sortilin luminal domains, provides an altered surface and the dimers sterically shield a large interface while bringing the two s-sortilin C-termini into close proximity. Biophysical and cell-based assays show that members of two different ligand families, (pro)neurotrophins and neurotensin, preferentially bind the sortilin monomer. This indicates that sortilin dimerization and conformational change discharges ligands and triggers recycling. More generally, this work may reveal a double mechanism for low pH-induced ligand release by endocytosis receptors.
TL;DR: Structural studies of the complex in the high spin and low spin state show that the strong cooperativity and thus the wide hysteresis is driven by an unprecedented anionic conformational change.
TL;DR: Using the enzyme from the industrial fungus Ashbya gossypii, it is demonstrated that the binding of adenine and guanine nucleotides to the canonical nucleotide binding sites of the regulatory Bateman domain induces different enzyme conformations with significantly distinct catalytic activities.
Abstract: Inosine-5'-monophosphate dehydrogenase (IMPDH) is an essential enzyme for nucleotide metabolism and cell proliferation. Despite IMPDH is the target of drugs with antiviral, immunosuppressive and antitumor activities, its physiological mechanisms of regulation remain largely unknown. Using the enzyme from the industrial fungus Ashbya gossypii, we demonstrate that the binding of adenine and guanine nucleotides to the canonical nucleotide binding sites of the regulatory Bateman domain induces different enzyme conformations with significantly distinct catalytic activities. Thereby, the comparison of their high-resolution structures defines the mechanistic and structural details of a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity of eukaryotic IMPDHs. Remarkably, retinopathy-associated mutations lie within the mechanical hinges of the conformational change, highlighting its physiological relevance. Our results expand the mechanistic repertoire of Bateman domains and pave the road to new approaches targeting IMPDHs.
TL;DR: Findings support an ADAM TS13 activation model in which VWF D4-CK engages the TSP8-CUB2 domains, inducing the conformational change that disrupts the CUB1-spacer domain interaction and thereby activates ADAMTS13.
TL;DR: Four residues at the α/β interface are identified and investigated of potential interest in kinetic gating of the E. coli Ia RNR machinery andutation of each residue resulted in loss of activity and with the exception of E52Q-β2, weakened subunit affinity.
TL;DR: Experimental and molecular modeling results showed that Cu(II) complex bound to DNA by a groove binding mode and induces detectable changes in the viscosity of DNA.
Abstract: The interaction of a new macrocyclic copper complex, [CuL(NO 3 ) 2 ] in which L is 1,3,6,10,12,15-hexaaza tricyclo[13.3.1.1 6,10 ] eicosane was investigated in vitro under simulated physiological conditions by multi-spectroscopic techniques and molecular modeling study. The fluorescence spectroscopy and UV absorption spectroscopy indicated the complex interacted with ct-DNA in a groove binding mode while the binding constant of UV–vis and the number of binding sites were 1.0 ± 0.2 × 10 4 L mol − 1 and 1.01, respectively. The fluorometric studies showed that the reaction between the complex with ct-DNA is exothermic (ΔH = 14.85 kJ mol − 1 ; ΔS = 109.54 J mol − 1 K − 1 ). Circular dichroism spectroscopy (CD) was employed to measure the conformational change of DNA in the presence of [CuL(NO 3 ) 2 ] complex. Furthermore, the complex induces detectable changes in the viscosity of DNA. The molecular modeling results illustrated that the complex strongly binds to groove of DNA. Experimental and molecular modeling results showed that Cu(II) complex bound to DNA by a groove binding mode.
TL;DR: Compared with the hole-hole homodimer under different storage conditions, the bispecific heterodimer, guided by the knob-into-hole assembly, proved to be a stable conformation with homogeneous distribution, confirming its high quality as a desired therapeutic.
Abstract: Bispecific antibodies have great potential to be the next-generation biotherapeutics due to their ability to simultaneously recognize two different targets. Compared to conventional monoclonal antibodies, knob-into-hole bispecific antibodies face unique challenges in production and characterization due to the increase in variant possibilities, such as homodimerization in covalent and noncovalent forms. In this study, a storage- and pH-sensitive hydrophobic interaction chromatography (HIC) profile change was observed for the hole-hole homodimer, and the multiple HIC peaks were explored and shown to be conformational isomers. We combined traditional analytical methods with hydrogen/deuterium exchange mass spectrometry (HDX MS), native mass spectrometry, and negative-staining electron microscopy to comprehensively characterize the hole-hole homodimer. HDX MS revealed conformational changes at the resolution of a few amino acids overlapping the CH2-CH3 domain interface. Conformational heterogeneity was also assessed by HDX MS isotopic distribution. The hole-hole homodimer was demonstrated to adopt a more homogeneous conformational distribution during storage. This conformational change is likely caused by a lack of CH3 domain dimerization (due to the three "hole" point mutations), resulting in a unique storage- and pH-dependent conformational destabilization and refolding of the hole-hole homodimer Fc. Compared with the hole-hole homodimer under different storage conditions, the bispecific heterodimer, guided by the knob-into-hole assembly, proved to be a stable conformation with homogeneous distribution, confirming its high quality as a desired therapeutic. Functional studies by antigen binding and neonatal Fc receptor (FcRn) binding correlated very well with the structural characterization. Comprehensive interpretation of the results has provided a better understanding of both the homodimer variant and the bispecific molecule.
TL;DR: The findings suggest that electrostatic interactions between domains II and IV in ERAP1 are crucial for driving a conformational change that regulates the structural integrity of the catalytic site.
Abstract: Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an intracellular enzyme that is important for the generation of antigenic epitopes and major histocompatibility class I-restricted adaptive immune responses. ERAP1 processes a vast variety of different peptides but still shows length and sequence selectivity, although the mechanism behind these properties is poorly understood. X-ray crystallographic analysis has revealed that ERAP1 can assume at least two distinct conformations in which C-terminal domain IV is either proximal or distal to active site domain II. To improve our understanding of the role of this conformational change in the catalytic mechanism of ERAP1, we used site-directed mutagenesis to perturb key salt bridges between domains II and IV. Enzymatic analysis revealed that these mutations, although located away from the catalytic site, greatly reduce the catalytic efficiency and change the allosteric kinetic behavior. The variants were more efficiently activated by small peptides and bound a ...
TL;DR: The overall results suggested that the PA‐based nanocarriers adopting the novel strategy of pH‐triggered secondary structural change could enhance therapeutic efficacy and be used as a promising platform for potential development of new generation of drug carriers for cancer therapy.