Deciphering the Multi-state Conformational Equilibrium of HDM2 in the Regulation of p53 Binding: Perspectives from Molecular Dynamics Simulation and NMR Analysis.

HDM2 negatively regulates the activity of the tumor suppressor p53. Previous NMR studies have shown that apo-HDM2 interconverts between an "open" state in which the N-terminal "lid" is disordered and a "closed" state in which the lid covers the p53-binding site in the core region. Molecular dynamics (MD) simulation studies have been performed to elucidate the conformational dynamics of HDM2, but the direct relevance of the experimental and computational analyses is unclear. In addition, how the phosphorylation of S17 in the lid contributes to the inhibition of p53 binding remains controversial. Here, we used both NMR and MD simulations to investigate the conformational dynamics of apo-HDM2. The NMR analysis revealed that apo-HDM2 exists in a fast-exchanging equilibrium within two closed states, closed 1 and closed 2, in addition to a previously demonstrated slow-exchanging "open-closed" equilibrium. MD simulations visualized two characteristic closed states, where the spatial orientation of the key residues corresponds well to the chemical shift changes of the NMR spectra. Furthermore, the phosphorylation of S17 induced an equilibrium shift toward closed 1, thereby suppressing the binding of p53 to HDM2. This study reveals a multi-state equilibrium of apo-HDM2 and provides new insights into the regulation mechanism of HDM2-p53 interactions.

Quantifying the Separation of Positive and Negative Areas in Electrostatic Potential for Predicting Feasibility of Ammonium Sulfate for Protein Crystallization.

Ammonium sulfate (AS) and poly(ethylene glycol) (PEG) are the most popular precipitants in protein crystallization. Some proteins are preferably crystallized by AS, while some are by PEG. The electrostatic potential is related to the preference of the precipitant agents. The iso-surfaces of the electrostatic potentials for the AS-crystallized proteins display a common shape and a distinct separation between the positive and negative areas. In contrast, the PEG-crystallized proteins show unclear positive and negative separation. In this work, we propose schemes to quantitatively evaluate the separation for predicting which precipitant is favorable for crystal growth between AS or PEG. Three methods were attempted to quantify the amplitude of the separation, separation distance, dipole moment, and shape regularity. The positive and negative areas are approximated to the spherical potentials caused by point charges. The first method is a measurement of the distance between the positive and negative point charges. The second one is an assessment including the quantity of electric charge into the distance. The last one is an approach monitoring the clarity of the positive and negative separation. The average value for 25 kinds of AS-preferring proteins was higher than that for the PEG-preferring ones in all three methods. Therefore, every method can distinguish the proteins preferring AS for crystal growth from those preferring PEG. These methods require an iso-surface of the electrostatic potential depicted at a certain contouring value. The shape of the iso-surface depends on the contouring value. The dependency on contour was examined by depicting the iso-surfaces of electrostatic potential with three values at ±0.8, ±0.5, and ±0.2 kT/e. While reducing the contouring value leads to the increase in separation distance and the decrease in shape regularity, dipole moment is independent of the alteration of contouring value. While the AS-preferring proteins are distinguishable from the PEG-preferring ones in any contouring values, the iso-surface at ±0.5 kT/e seems adequate for regular use. The dipole moment assessment is feasible for the choice of potent precipitants for crystal growth in experiments.

Analysis of Binding Modes of Antigen-Antibody Complexes by Molecular Mechanics Calculation.

Antibodies are one of the most important protein molecules in biopharmaceutics. Due to the recent advance in technology for producing monoclonal antibodies, many structural data are available on the antigen-antibody complexes. To characterize the molecular interaction in antigen-antibody recognition, we computationally analyzed 500 complex structures by molecular mechanics calculations. The presence of Ser and Tyr is markedly large in the complementarity-determining regions (CDRs). Although Ser is abundant in CDRs, its contribution to the binding score is not large. Instead, Tyr, Asp, Glu, and Arg significantly contribute to the molecular interaction from the viewpoint of the binding score. The decomposition of the binding score suggests that the hydrophilic interaction is predominant in all CDRs compared with the hydrophobic one. The contribution of the heavy chain is larger than that of the light chain. In particular, H2 and H3 largely contribute to the binding interaction. Tyr is a main contributing residue both in H2 and H3. The positively charged residue Arg also significantly contributes to the binding score in H3, while the contribution of Lys is small. The appearance of Ser is remarkable in H2, and Asp is abundant in H3. The non-charged polar residues, Thr, Asn, and Gln, appear much in H2, compared to appearing in H3. The negatively charged residues Asp and Glu significantly contribute to the binding score in H3. The contributions of Phe and Trp are not large in spite that the aromatic residues are capable of making the π-π or CH-π interaction. Gly is commonly abundant both in H2 and H3. The average distance of the shortest direct hydrogen bond between the antigen and antibody is longer than that of the hydrogen bonds observed in the complexes between compounds and their target proteins. Therefore, the antigen-antibody interface is not so tight as the compound-target protein interface. The calculation of shape complementarity is consistent with the result of the hydrogen bonds in that the fitness of the antigen-antibody contact is not so high as that of the compound-target protein contact. There exist many water molecules at the antigen-antibody interface. These findings suggest that Tyr, Asp, Glu, and Arg are rich in H3 and work as major contributors for the interaction with the antigen. Ser, Thr, Asn, and Gln are rich in H2 and support the interaction with enhancing molecular fitness. Gly is helpful in increasing flexibility and geometrical diversity. Because the antigen-antibody binding is fundamentally hydrophilic-driven, the non-polar residues are unfavorable for mediating the contact even for the aromatic residues such as Phe and Trp.

Balanced Regulation of Redox Status of Intracellular Thioredoxin Revealed by in-Cell NMR.

To understand how intracellular proteins respond to oxidative stresses, the redox status of the target protein, as well as the intracellular redox potential ( EGSH), which is defined by the concentrations of reduced and oxidized glutathione, should be observed simultaneously within living cells. In this study, we developed a method that can monitor the redox status of thioredoxin (Trx) and EGSH by direct NMR observation of Trx and glutathione within living cells. Unlike the midpoint potential of Trx measured in vitro (∼ -300 mV), the intracellular Trx exhibited the redox transition at EGSH between -250 and -200 mV, the range known to trigger the oxidative stress-mediated signalings. Furthermore, we quantified the contribution of Trx reductase to the redox status of Trx, demonstrating that the redox profile of Trx is determined by the interplay between the elevation of EGSH and the reduction by Trx reductase and other endogenous molecules.

Mechanical force effect on the two-state equilibrium of the hyaluronan-binding domain of CD44 in cell rolling.

CD44 is the receptor for hyaluronan (HA) and mediates cell rolling under fluid shear stress. The HA-binding domain (HABD) of CD44 interconverts between a low-affinity, ordered (O) state and a high-affinity, partially disordered (PD) state, by the conformational change of the C-terminal region, which is connected to the plasma membrane. To examine the role of tensile force on CD44-mediated rolling, we used a cell-free rolling system, in which recombinant HABDs were attached to beads through a C-terminal or N-terminal tag. We found that the rolling behavior was stabilized only at high shear stress, when the HABD was attached through the C-terminal tag. In contrast, no difference was observed for the beads coated with HABD mutants that constitutively adopt either the O state or the PD state. Steered molecular dynamics simulations suggested that the force from the C terminus disrupts the interaction between the C-terminal region and the core of the domain, thus providing structural insights into how the mechanical force triggers the allosteric O-to-PD transition. Based on these results, we propose that the force applied from the C terminus enhances the HABD-HA interactions by inducing the conformational change to the high-affinity PD transition more rapidly, thereby enabling CD44 to mediate lymphocyte trafficking and hematopoietic progenitor cell homing under high-shear conditions.

Cross-saturation and transferred cross-saturation experiments.

Structural analyses of protein-protein interactions are required to reveal their functional mechanisms, and accurate protein-protein complex models, based on experimental results, are the starting points for drug development. In addition, structural information about proteins under physiologically relevant conditions is crucially important for understanding biological events. However, for proteins such as those embedded in lipid bilayers and transiently complexed with their effectors under physiological conditions, structural analyses by conventional methods are generally difficult, due to their large molecular weights and inhomogeneity. We have developed the cross-saturation (CS) method, which is an nuclear magnetic resonance measurement technique for the precise identification of the interfaces of protein-protein complexes. In addition, we have developed an extended version of the CS method, termed transferred cross-saturation (TCS), which enables the identification of the residues of protein ligands in close proximity to huge (>150 kDa) and heterogeneous complexes under fast exchange conditions (>0.1 s(-1)). Here, we discuss the outline, basic theory, and practical considerations of the CS and TCS methods. In addition, we will review the recent progress in the construction of models of protein-protein complexes, based on CS and TCS experiments, and applications of TCS to in situ analyses of biologically and medically important proteins in physiologically relevant states.

Structure of a complete integrin ectodomain in a physiologic resting state and activation and deactivation by applied forces.

The complete ectodomain of integrin alpha(IIb)beta(3) reveals a bent, closed, low-affinity conformation, the beta knee, and a mechanism for linking cytoskeleton attachment to high affinity for ligand. Ca and Mg ions in the recognition site, including the synergistic metal ion binding site (SyMBS), are loaded prior to ligand binding. Electrophilicity of the ligand-binding Mg ion is increased in the open conformation. The beta(3) knee passes between the beta(3)-PSI and alpha(IIb)-knob to bury the lower beta leg in a cleft, from which it is released for extension. Different integrin molecules in crystals and EM reveal breathing that appears on pathway to extension. Tensile force applied to the extended ligand-receptor complex stabilizes the closed, low-affinity conformation. By contrast, an additional lateral force applied to the beta subunit to mimic attachment to moving actin filaments stabilizes the open, high-affinity conformation. This mechanism propagates allostery over long distances and couples cytoskeleton attachment of integrins to their high-affinity state.

A pH-regulated dimeric bouquet in the structure of von Willebrand factor.

At the acidic pH of the trans-Golgi and Weibel-Palade bodies (WPBs), but not at the alkaline pH of secretion, the C-terminal ∼1350 residues of von Willebrand factor (VWF) zip up into an elongated, dimeric bouquet. Six small domains visualized here for the first time between the D4 and cystine-knot domains form a stem. The A2, A3, and D4 domains form a raceme with three pairs of opposed, large, flower-like domains. N-terminal VWF domains mediate helical tubule formation in WPBs and template N-terminal disulphide linkage between VWF dimers, to form ultralong VWF concatamers. The dimensions we measure in VWF at pH 6.2 and 7.4, and the distance between tubules in nascent WPB, suggest that dimeric bouquets are essential for correct VWF dimer incorporation into growing tubules and to prevent crosslinking between neighbouring tubules. Further insights into the structure of the domains and flexible segments in VWF provide an overall view of VWF structure important for understanding both the biogenesis of ultralong concatamers at acidic pH and flow-regulated changes in concatamer conformation in plasma at alkaline pH that trigger hemostasis.

Structure of an integrin with an alphaI domain, complement receptor type 4.

We report the structure of an integrin with an alphaI domain, alpha(X)beta(2), the complement receptor type 4. It was earlier expected that a fixed orientation between the alphaI domain and the beta-propeller domain in which it is inserted would be required for allosteric signal transmission. However, the alphaI domain is highly flexible, enabling two betaI domain conformational states to couple to three alphaI domain states, and greater accessibility for ligand recognition. Although alpha(X)beta(2) is bent similarly to integrins that lack alphaI domains, the terminal domains of the alpha- and beta-legs, calf-2 and beta-tail, are oriented differently than in alphaI-less integrins. Linkers extending to the transmembrane domains are unstructured. Previous mutations in the beta(2)-tail domain support the importance of extension, rather than a deadbolt, in integrin activation. The locations of further activating mutations and antibody epitopes show the critical role of extension, and conversion from the closed to the open headpiece conformation, in integrin activation. Differences among 10 molecules in crystal lattices provide unprecedented information on interdomain flexibility important for modelling integrin extension and activation.

Real-Time Monitoring of RAS Activity Using In Vitro and In-Cell NMR Spectroscopy.

The activation level of RAS can be determined by GTP hydrolysis rate (khy) and GDP-GTP exchange rates (kex). Either impaired GTP hydrolysis or enhanced GDP-GTP exchange causes the aberrant activation of RAS in oncogenic mutants. Therefore, it is important to quantify the khy and kex for understanding the mechanisms of RAS oncogenesis and drug development. Conventional methods have individually measured the kex and khy of RAS. However, within the intracellular environment, GTP hydrolysis and GDP-GTP exchange reactions occur simultaneously under conditions where GTP concentration is kept constant. In addition, the intracellular activity of RAS is influenced by endogenous regulatory proteins, such as RAS GTPase activating proteins (GAPs) and the guanine-nucleotide exchange factors (GEFs). Here, we describe the in vitro and in-cell NMR methods to estimate the khy and kex simultaneously by measuring the time-dependent changes of the fraction of GTP-bound ratio under the condition of constant GTP concentration.

Requirement of open headpiece conformation for activation of leukocyte integrin alphaXbeta2.

Negative stain electron microscopy (EM) and adhesion assays show that alpha(X)beta(2) integrin activation requires headpiece opening as well as extension. An extension-inducing Fab to the beta(2) leg, in combination with representative activating and inhibitory Fabs, were examined for effect on the equilibrium between the open and closed headpiece conformations. The two activating Fabs stabilized the open headpiece conformation. Conversely, two different inhibitory Fabs stabilized the closed headpiece conformation. Adhesion assays revealed that alpha(X)beta(2) in the extended-open headpiece conformation had high affinity for ligand, whereas both the bent conformation and the extended-closed headpiece conformation represented the low affinity state. Intermediate integrin affinity appears to result not from a single conformational state, but from a mixture of equilibrating conformational states.

Real-time monitoring of the reaction of KRAS G12C mutant specific covalent inhibitor by in vitro and in-cell NMR spectroscopy.

KRAS mutations are major drivers of various cancers. Recently, allele-specific inhibitors of the KRAS G12C mutant were developed that covalently modify the thiol of Cys12, thereby trapping KRAS in an inactive GDP-bound state. To study the mechanism of action of the covalent inhibitors in both in vitro and intracellular environments, we used real-time NMR to simultaneously observe GTP hydrolysis and inhibitor binding. In vitro NMR experiments showed that the rate constant of ARS-853 modification is identical to that of GTP hydrolysis, indicating that GTP hydrolysis is the rate-limiting step for ARS-853 modification. In-cell NMR analysis revealed that the ARS-853 reaction proceeds significantly faster than that in vitro, reflecting acceleration of GTP hydrolysis by endogenous GTPase proteins. This study demonstrated that the KRAS covalent inhibitor is as effective in the cell as in vitro and that in-cell NMR is a valuable validation tool for assessing the pharmacological properties of the drug in the intracellular context.

Nanoscopic Elucidation of Spontaneous Self-Assembly of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Open Reading Frame 6 (ORF6) Protein.

Open reading frame 6 (ORF6), the accessory protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that suppresses host type-I interferon signaling, possesses amyloidogenic sequences. ORF6 amyloidogenic peptides self-assemble to produce cytotoxic amyloid fibrils. Currently, the molecular properties of the ORF6 remain elusive. Here, we investigate the structural dynamics of the full-length ORF6 protein in a near-physiological environment using high-speed atomic force microscopy. ORF6 oligomers were ellipsoidal and readily assembled into ORF6 protofilaments in either a circular or a linear pattern. The formation of ORF6 protofilaments was enhanced at higher temperatures or on a lipid substrate. ORF6 filaments were sensitive to aliphatic alcohols, urea, and SDS, indicating that the filaments were predominantly maintained by hydrophobic interactions. In summary, ORF6 self-assembly could be necessary to sequester host factors and causes collateral damage to cells via amyloid aggregates. Nanoscopic imaging unveiled the innate molecular behavior of ORF6 and provides insight into drug repurposing to treat amyloid-related coronavirus disease 2019 complications.

In situ structural biology using in-cell NMR.

BACKGROUND: Accumulating evidence from the experimental and computational studies indicated that the functional properties of proteins are different between in vitro and living cells, raising the necessity to examine the protein structure under the native intracellular milieu. To gain structural information of the proteins inside the living cells at an atomic resolution, in-cell NMR method has been developed for the past two decades. SCOPE OF REVIEW: In this review, we will overview the recent progress in the methodological developments and the biological applications of in-cell NMR, and discuss the advances and challenges in this filed. MAJOR CONCLUSIONS: A number of methods were developed to enrich the isotope-labeled proteins inside the cells, enabling the in-cell NMR observation of bacterial cells as well as eukaryotic cells. In-cell NMR has been applied to various biological systems, including de novo structure determinations, protein/protein or protein/drug interactions, and monitoring of chemical reactions exerted by the endogenous enzymes. The bioreactor system, in which the cells in the NMR tube are perfused by fresh culture medium, enabled the long-term in-cell NMR measurements, and the real-time observations of intracellular responses upon external stimuli. GENERAL SIGNIFICANCE: In-cell NMR has become a unique technology for its ability to obtain the function-related structural information of the target proteins under the physiological or pathological cellular environments, which cannot be reconstituted in vitro.

Real-Time In-Cell NMR Reveals the Intracellular Modulation of GTP-Bound Levels of RAS.

The small guanosine triphosphatase (GTPase) RAS serves as a molecular switch in signal transduction, and its mutation and aberrant activation are implicated in tumorigenesis. Here, we perform real-time, in-cell nuclear magnetic resonance (NMR) analyses of non-farnesylated RAS to measure time courses of the fraction of the active GTP-bound form (fGTP) within cytosol of live mammalian cells. The observed intracellular fGTP is significantly lower than that measured in vitro for wild-type RAS as well as oncogenic mutants, due to both decrease of the guanosine diphosphate (GDP)-GTP exchange rate (kex) and increase of GTP hydrolysis rate (khy). In vitro reconstitution experiments show that highly viscous environments promote a reduction of kex, whereas the increase of khy is stimulated by unidentified cytosolic proteins. This study demonstrates the power of in-cell NMR to directly detect the GTP-bound levels of RAS in mammalian cells, thereby revealing that the khy and kex of RAS are modulated by various intracellular factors.

Structural basis for two-way communication between dynein and microtubules.

The movements of cytoplasmic dynein on microtubule (MT) tracks is achieved by two-way communication between the microtubule-binding domain (MTBD) and the ATPase domain via a coiled-coil stalk, but the structural basis of this communication remains elusive. Here, we regulate MTBD either in high-affinity or low-affinity states by introducing a disulfide bond to the stalk and analyze the resulting structures by NMR and cryo-EM. In the MT-unbound state, the affinity changes of MTBD are achieved by sliding of the stalk α-helix by a half-turn, which suggests that structural changes propagate from the ATPase-domain to MTBD. In addition, MT binding induces further sliding of the stalk α-helix even without the disulfide bond, suggesting how the MT-induced conformational changes propagate toward the ATPase domain. Based on differences in the MT-binding surface between the high- and low-affinity states, we propose a potential mechanism for the directional bias of dynein movement on MT tracks.

Observation of NMR signals from proteins introduced into living mammalian cells by reversible membrane permeabilization using a pore-forming toxin, streptolysin O.

We have developed a new in-cell NMR method that is applicable to any type of cell and does not require target protein modification or specialized equipment. The stable-isotope-labeled target protein, thymosin beta4 (Tbeta4), was delivered to 293F cells, which were permeabilized by a pore-forming toxin, streptolysin O, and resealed by Ca(2+) after Tbeta4 uptake. As a result, we successfully observed (1)H-(15)N HSQC signals originating from the Tbeta4, including those from the N-terminal acetylation, which had occurred inside the cell as a post-translational modification.

Functional and structural stability of the epidermal growth factor receptor in detergent micelles and phospholipid nanodiscs.

Cellular signaling mediated by the epidermal growth factor receptor (EGFR or ErbB) family of receptor tyrosine kinases plays an important role in regulating normal and oncogenic cellular physiology. While structures of isolated EGFR extracellular domains and intracellular protein tyrosine kinase domains have suggested mechanisms for growth factor-mediated receptor dimerization and allosteric kinase domain activation, understanding how the transmembrane and juxtamembrane domains contribute to transmembrane signaling requires structural studies on intact receptor molecules. In this report, recombinant EGFR constructs containing the extracellular, transmembrane, juxtamembrane, and kinase domains are overexpressed and purified from human embryonic kidney 293 cell cultures. The oligomerization state, overall structure, and functional stability of the purified EGF-bound receptor are characterized in detergent micelles and phospholipid bilayers. In the presence of EGF, catalytically active EGFR dimers can be isolated by gel filtration in dodecyl maltoside. Visualization of the dimeric species by negative stain electron microscopy and single particle averaging reveals an overall structure of the extracellular domain that is similar to previously published crystal structures and is consistent with the C-termini of domain IV being juxtaposed against one another as they enter the transmembrane domain. Although detergent-soluble preparations of EGFR are stable as dimers in the presence of EGF, they exhibit differential functional stability in Triton X-100 versus dodecyl maltoside. Furthermore, the kinase activity can be significantly stabilized by reconstituting purified EGF-bound EGFR dimers in phospholipid nanodiscs or vesicles, suggesting that the environment around the hydrophobic transmembrane and amphipathic juxtamembrane domains is important for stabilizing the tyrosine kinase activity in vitro.

Probing dynamics and conformational change of the GroEL-GroES complex by 13C NMR spectroscopy.

Bacterial chaperonin GroEL with a molecular mass of 800 kDa was studied by (13)C NMR spectroscopy. Carbonyl carbons of GroEL were labeled with (13)C in an amino acid specific manner in order to reduce the number of signals to be observed in the spectrum. Combination of selective labeling and site-directed mutagenesis enabled us to establish the sequence specific assignment of the (13)C resonances from GroEL. ADP-binding induced a chemical shift change of Tyr478 in the equatorial domain and His401 in the intermediate domain, but little of Tyr203 in the apical domain. Upon complex formation with co-chaperonin GroES in the presence of ADP, Tyr478 exhibits two peaks that would originate from the cis and trans rings of the asymmetric GroEL-GroES complex. Comparison between the line width of the GroEL resonances and those from GroES in complex with GroEL revealed broadening disproportionate to the size of GroEL, implying the existence of conformational fluctuations which may be pertinent to the chaperone activity. Based on these results, we concluded that (13)C NMR observation in combination with selective labeling and site-directed mutagenesis can be utilized for probing the conformational change and dynamics of the extremely large molecules that are inaccessible with current NMR methods.