Residue-based correlation between equilibrium and rate constants is an experimental formulation of the consistency principle for smooth structural changes of proteins.

The consistency principle represents a physicochemical condition requisite for ideal protein folding. It assumes that any pair of amino acid residues in partially folded structures has an attractive short-range interaction only if the two residues are in contact within the native structure. The residue-specific equilibrium constant, K, and the residue-specific rate constant, k (forward and backward), can be determined by NMR and hydrogen-deuterium exchange studies. Linear free energy relationships (LFER) in the rate-equilibrium free energy relationship (REFER) plots (i.e., log k vs. log K) are widely seen in protein-related phenomena, but our REFER plot differs from them in that the data points are derived from one polypeptide chain under a single condition. Here, we examined the theoretical basis of the residue-based LFER. First, we derived a basic equation, ρij=½(φi+φj), from the consistency principle, where ρij is the slope of the line segment that connects residues i and j in the REFER plot, and φi and φj are the local fractions of the native state in the transient state ensemble (TSE). Next, we showed that the general solution is the alignment of the (log K, log k) data points on a parabolic curve in the REFER plot. Importantly, unlike LFER, the quadratic free energy relationship (QFER) is compatible with the heterogeneous formation of local structures in the TSE. Residue-based LFER/QFER provides a unique insight into the TSE: A foldable polypeptide chain consists of several folding units, which are consistently coupled to undergo smooth structural changes.

Retrospective study for the universal applicability of the residue-based linear free energy relationship in the two-state exchange of protein molecules.

Multiprobe measurements, such as NMR and hydrogen exchange studies, can provide the equilibrium constant, K, and rate constants for forward and backward processes, k and k', of the two-state structural changes of a polypeptide on a per-residue basis. We previously found a linear relationship between log K and log k and between log K and log k' for the topological exchange of a 27-residue bioactive peptide. To test the general applicability of the residue-based linear free energy relationship (rbLEFR), we performed a literature search to collect residue-specific K, k, and k' values in various exchange processes, including folding-unfolding equilibrium, coupled folding and binding of intrinsically disordered peptides, and structural fluctuations of folded proteins. The good linearity in a substantial number of the log-log plots proved that the rbLFER holds for the structural changes in a wide variety of protein-related phenomena. Among the successful cases, the hydrogen exchange study of apomyoglobin folding intermediates is particularly interesting. We found that the residues that deviated from the linear relationship corresponded to the α-helix, for which transient translocation had been identified by other experiments. Thus, the rbLFER is useful for studying the structures and energetics of the dynamic states of protein molecules.

The time-zero HSQC method improves the linear free energy relationship of a polypeptide chain through the accurate measurement of residue-specific equilibrium constants.

EXSY (exchange spectroscopy) NMR provides the residue-specific equilibrium constants, K, and residue-specific kinetic rate constants, k, of a polypeptide chain in a two-state exchange in the slow exchange regime. A linear free energy relationship (LFER) discovered in a log k versus log K plot is considered to be a physicochemical basis for smooth folding and conformational changes of protein molecules. For accurate determination of the thermodynamic and kinetic parameters, the measurement bias arising from state-specific differences in the R1 and R2 relaxation rates of 1H and other nuclei in HSQC and EXSY experiments must be minimized. Here, we showed that the time-zero HSQC acquisition scheme (HSQC0) is effective for this purpose, in combination with a special analytical method (Π analysis) for EXSY. As an example, we applied the HSQC0 + Π method to the two-state exchange of nukacin ISK-1 in an aqueous solution. Nukacin ISK-1 is a 27-residue lantibiotic peptide containing three mono-sulfide linkages. The resultant bias-free residue-based LFER provided valuable insights into the transition state of the topological interconversion of nukacin ISK-1. We found that two amino acid residues were exceptions in the residue-based LFER relationship. We inferred that the two residues could adopt special conformations in the transition state, to allow the threading of some side chains through a ring structure formed by one of the mono-sulfide linkages. In this context, the two residues are a useful target for the manipulation of the physicochemical properties and biological activities of nukacin ISK-1.

Crystal structure of the PX domain of Vps17p from Saccharomyces cerevisiae.

The structure determination of the PX (phox homology) domain of the Saccharomyces cerevisiae Vps17p protein presented a challenging case for molecular replacement because it has noncrystallographic symmetry close to a crystallographic axis. The combination of diffraction-quality crystals grown under microgravity on the International Space Station and a highly accurate template structure predicted by AlphaFold2 provided the key to successful crystal structure determination. Although the structure of the Vps17p PX domain is seen in many PX domains, no basic residues are found around the canonical phosphatidylinositol phosphate (PtdIns-P) binding site, suggesting an inability to bind PtdIns-P molecules.

Residue-Specific Kinetic Insights into the Transition State in Slow Polypeptide Topological Isomerization by NMR Exchange Spectroscopy.

The characterization of the transition state is a central issue in biophysical studies of protein folding. NMR is a multiprobe measurement technique that provides residue-specific information. Here, we used exchange spectroscopy to characterize the transition state of the two-state slow topological isomerization of a 27-residue lantibiotic peptide. The exchange kinetic rates varied on a per-residue basis, indicating the reduced kinetic cooperativity of the two-state exchange, as well as the previously observed reduced thermodynamic cooperativity. Furthermore, temperature-dependent measurements revealed large variations in the activation enthalpy and entropy terms among residues. Interestingly, we found a linear relationship between the logarithm of the equilibrium constants and that of the exchange rates. Because the data points are derived from amino acid residues in one polypeptide chain, we refer to the linear relationship as the residue-based linear free energy relationship (rbLFER). The rbLFER offers information about the transition state of the two-state exchange.

The benzylisoquinoline alkaloids, berberine and coptisine, act against camptothecin-resistant topoisomerase I mutants.

DNA replication inhibitors are utilized extensively in studies of molecular biology and as chemotherapy agents in clinical settings. The inhibition of DNA replication often triggers double-stranded DNA breaks (DSBs) at stalled DNA replication sites, resulting in cytotoxicity. In East Asia, some traditional medicines are administered as anticancer drugs, although the mechanisms underlying their pharmacological effects are not entirely understood. In this study, we screened Japanese herbal medicines and identified two benzylisoquinoline alkaloids (BIAs), berberine and coptisine. These alkaloids mildly induced DSBs, and this effect was dependent on the function of topoisomerase I (Topo I) and MUS81-EME1 structure-specific endonuclease. Biochemical analysis revealed that the action of BIAs involves inhibiting the catalytic activity of Topo I rather than inducing the accumulation of the Topo I-DNA complex, which is different from the action of camptothecin (CPT). Furthermore, the results showed that BIAs can act as inhibitors of Topo I, even against CPT-resistant mutants, and that the action of these BIAs was independent of CPT. These results suggest that using a combination of BIAs and CPT might increase their efficiency in eliminating cancer cells.

Two-State Exchange Dynamics in Membrane-Embedded Oligosaccharyltransferase Observed in Real-Time by High-Speed AFM.

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of oligosaccharide chains from lipid-linked oligosaccharides (LLO) to asparagine residues in polypeptide chains. Using high-speed atomic force microscopy (AFM), we investigated the dynamic properties of OST molecules embedded in biomembranes. An archaeal single-subunit OST protein was immobilized on a mica support via biotin-avidin interactions and reconstituted in a lipid bilayer. The distance between the top of the protein molecule and the upper surface of the lipid bilayer was monitored in real-time. The height of the extramembranous part exhibited a two-step variation with a difference of 1.8 nm. The high and low states are designated as state 1 and state 2, respectively. The transition processes between the two states fit well to single exponential functions, suggesting that the observed dynamic exchange is an intrinsic property of the archaeal OST protein. The two sets of cross peaks in the NMR spectra of the protein supported the conformational changes between the two states in detergent-solubilized conditions. Considering the height values measured in the AFM measurements, state 1 is closer to the crystal structure, and state 2 has a more compact form. Subsequent AFM experiments indicated that the binding of the sugar donor LLO decreased the structural fluctuation and shifted the equilibrium almost completely to state 1. This dynamic behavior is likely necessary for efficient catalytic turnover. Presumably, state 2 facilitates the immediate release of the bulky glycosylated polypeptide product, thus allowing OST to quickly prepare for the next catalytic cycle.

Mosaic Cooperativity in Slow Polypeptide Topological Isomerization Revealed by Residue-Specific NMR Thermodynamic Analysis.

Slow polypeptide conformational changes on time scales of >1 s are generally assumed to be highly cooperative two-state transitions, reflecting the high energy barrier. However, few experimental characterizations have tested the validity of this assumption. We performed residue-specific NMR thermodynamic analysis of the 27-residue lantibiotic peptide, nukacin ISK-1, to characterize the isomerization between two topological states on the second time scale. Unexpectedly, the thermal transition behaviors were distinct among peptide regions, indicating that the topological isomerization process is a mosaic of different degrees of cooperativity. The conformational change path between the two NMR structures was deduced by a targeted molecular dynamics simulation. The unique side-chain threading motions through the monosulfide rings are the structural basis of the high energy barrier, and the nonlocal interactions in the hydrophobic core are the structural basis of the cooperativity. Taken together, we provide an energetic description of the topological isomerization of nukacin ISK-1.

Phagocytosis is mediated by two-dimensional assemblies of the F-BAR protein GAS7.

Phagocytosis is a cellular process for internalization of micron-sized large particles including pathogens. The Bin-Amphiphysin-Rvs167 (BAR) domain proteins, including the FCH-BAR (F-BAR) domain proteins, impose specific morphologies on lipid membranes. Most BAR domain proteins are thought to form membrane invaginations or protrusions by assembling into helical submicron-diameter filaments, such as on clathrin-coated pits, caveolae, and filopodia. However, the mechanism by which BAR domain proteins assemble into micron-scale phagocytic cups was unclear. Here, we show that the two-dimensional sheet-like assembly of Growth Arrest-Specific 7 (GAS7) plays a critical role in phagocytic cup formation in macrophages. GAS7 has the F-BAR domain that possesses unique hydrophilic loops for two-dimensional sheet formation on flat membranes. Super-resolution microscopy reveals the similar assemblies of GAS7 on phagocytic cups and liposomes. The mutations of the loops abolishes both the membrane localization of GAS7 and phagocytosis. Thus, the sheet-like assembly of GAS7 plays a significant role in phagocytosis.

Structural Basis of Protein Asn-Glycosylation by Oligosaccharyltransferases.

Glycosylation of asparagine residues is a ubiquitous protein modification. This N-glycosylation is essential in Eukaryotes, but principally nonessential in Prokaryotes (Archaea and Eubacteria), although it facilitates their survival and pathogenicity. In many reviews, Archaea have received far less attention than Eubacteria, but this review will cover the N-glycosylation in the three domains of life. The oligosaccharide chain is preassembled on a lipid-phospho carrier to form a donor substrate, lipid-linked oligosaccharide (LLO). The en bloc transfer of an oligosaccharide from LLO to selected Asn residues in the Asn-X-Ser/Thr (X≠Pro) sequons in a polypeptide chain is catalyzed by a membrane-bound enzyme, oligosaccharyltransferase (OST). Over the last 10 years, the three-dimensional structures of the catalytic subunits of the Stt3/AglB/PglB proteins, with an acceptor peptide and a donor LLO, have been determined by X-ray crystallography, and recently the complex structures with other subunits have been determined by cryo-electron microscopy . Structural comparisons within the same species and across the different domains of life yielded a unified view of the structures and functions of OSTs. A catalytic structure in the TM region accounts for the amide bond twisting, which increases the reactivity of the side-chain nitrogen atom of the acceptor Asn residue in the sequon. The Ser/Thr-binding pocket in the C-terminal domain explains the requirement for hydroxy amino acid residues in the sequon. As expected, the two functional structures are formed by the involvement of short amino acid motifs conserved across the three domains of life.

Structures of the prefusion form of measles virus fusion protein in complex with inhibitors.

Measles virus (MeV), a major cause of childhood morbidity and mortality, is highly immunotropic and one of the most contagious pathogens. MeV may establish, albeit rarely, persistent infection in the central nervous system, causing fatal and intractable neurodegenerative diseases such as subacute sclerosing panencephalitis and measles inclusion body encephalitis. Recent studies have suggested that particular substitutions in the MeV fusion (F) protein are involved in the pathogenesis by destabilizing the F protein and endowing it with hyperfusogenicity. Here we show the crystal structures of the prefusion MeV-F alone and in complex with the small compound AS-48 or a fusion inhibitor peptide. Notably, these independently developed inhibitors bind the same hydrophobic pocket located at the region connecting the head and stalk of MeV-F, where a number of substitutions in MeV isolates from neurodegenerative diseases are also localized. Since these inhibitors could suppress membrane fusion mediated by most of the hyperfusogenic MeV-F mutants, the development of more effective inhibitors based on the structures may be warranted to treat MeV-induced neurodegenerative diseases.

Tethering an N-Glycosylation Sequon-Containing Peptide Creates a Catalytically Competent Oligosaccharyltransferase Complex.

Oligosaccharyltransferase (OST) transfers an oligosaccharide chain to the Asn residue in the Asn-X-Ser/Thr sequon in proteins, where X is not proline. A sequon was tethered to an archaeal OST enzyme via a disulfide bond. The positions of the cysteine residues in the OST protein and the sequon-containing acceptor peptide were selected by reference to the eubacterial OST structure in a noncovalent complex with an acceptor peptide. We determined the crystal structure of the cross-linked OST-sequon complex. The Ser/Thr-binding pocket recognizes the Thr residue in the sequon, and the catalytic structure termed the "carboxylate dyad" interacted with the Asn residue. Thus, the recognition and the catalytic mechanism of the sequon are conserved between the archaeal and eubacterial OSTs. We found that the tethered peptides in the complex were efficiently glycosylated in the presence of the oligosaccharide donor. The stringent requirements are greatly relaxed in the cross-linked state. The two conserved acidic residues in the catalytic structure were each dispensable, although the double mutation abolished the activity. A Gln residue at the Asn position in the sequon functioned as an acceptor, and the hydroxy group at position +2 was not required. In the standard assay using short free peptides, strong amino acid preferences were observed at the X position, but the preferences, except for Pro, completely disappeared in the cross-linked state. By skipping the initial binding process and stabilizing the complex state, the catalytically competent cross-linked complex offers a unique system for studying the oligosaccharyl transfer reaction.

Comparative Analysis of Archaeal Lipid-linked Oligosaccharides That Serve as Oligosaccharide Donors for Asn Glycosylation.

The glycosylation of asparagine residues is the predominant protein modification in all three domains of life. An oligosaccharide chain is preassembled on a lipid-phospho carrier and transferred onto asparagine residues by the action of a membrane-bound enzyme, oligosaccharyltransferase. The oligosaccharide donor for the oligosaccharyl transfer reaction is dolichol-diphosphate-oligosaccharide in Eukaryota and polyprenol-diphosphate-oligosaccharide in Eubacteria. The donor in some archaeal species was reportedly dolichol-monophosphate-oligosaccharide. Thus, the difference in the number of phosphate groups aroused interest in whether the use of the dolichol-monophosphate type donors is widespread in the domain Archaea. Currently, all of the archaeal species with identified oligosaccharide donors have belonged to the phylum Euryarchaeota. Here, we analyzed the donor structures of two species belonging to the phylum Crenarchaeota, Pyrobaculum calidifontis and Sulfolobus solfataricus, in addition to two species from the Euryarchaeota, Pyrococcus furiosus and Archaeoglobus fulgidus The electrospray ionization tandem mass spectrometry analyses confirmed that the two euryarchaeal oligosaccharide donors were the dolichol-monophosphate type and newly revealed that the two crenarchaeal oligosaccharide donors were the dolichol-diphosphate type. This novel finding is consistent with the hypothesis that the ancestor of Eukaryota is rooted within the TACK (Thaum-, Aig-, Cren-, and Korarchaeota) superphylum, which includes Crenarchaea. Our comprehensive study also revealed that one archaeal species could contain two distinct oligosaccharide donors for the oligosaccharyl transfer reaction. The A. fulgidus cells contained two oligosaccharide donors bearing oligosaccharide moieties with different backbone structures, and the S. solfataricus cells contained two oligosaccharide donors bearing stereochemically different dolichol chains.

Rational design of crystal contact-free space in protein crystals for analyzing spatial distribution of motions within protein molecules.

Contacts with neighboring molecules in protein crystals inevitably restrict the internal motions of intrinsically flexible proteins. The resultant clear electron densities permit model building, as crystallographic snapshot structures. Although these still images are informative, they could provide biased pictures of the protein motions. If the mobile parts are located at a site lacking direct contacts in rationally designed crystals, then the amplitude of the movements can be experimentally analyzed. We propose a fusion protein method, to create crystal contact-free space (CCFS) in protein crystals and to place the mobile parts in the CCFS. Conventional model building fails when large amplitude motions exist. In this study, the mobile parts appear as smeared electron densities in the CCFS, by suitable processing of the X-ray diffraction data. We applied the CCFS method to a highly mobile presequence peptide bound to the mitochondrial import receptor, Tom20, and a catalytically relevant flexible segment in the oligosaccharyltransferase, AglB. These two examples demonstrated the general applicability of the CCFS method to the analysis of the spatial distribution of motions within protein molecules.

Structural basis for Marburg virus neutralization by a cross-reactive human antibody.

The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.

The BALB/c-specific polymorphic SIRPA enhances its affinity for human CD47, inhibiting phagocytosis against human cells to promote xenogeneic engraftment.

It has been shown that in xenotransplantation of human cells into immunodeficient mice, the mouse strain background is critical. For example, the nonobese diabetic (NOD) strain is most efficient, the BALB/c is moderate, and the C57BL/6 is inefficient for human cell engraftment. We have shown that the NOD-specific polymorphism of the signal regulatory protein-alpha (Sirpa) allows NOD SIRPA to bind human CD47, and the resultant "don't eat me" signaling by this binding prevents host macrophages to engulf human grafts, thereby inhibiting rejection. Here we tested whether the efficient xenotransplantation capability of the BALB/c strain is also mediated by the SIRPA-CD47 self-recognition system. BALB/c SIRPA was capable of binding to human CD47 at an intermediate level between those of C57BL/6 SIRPA and NOD SIRPA. Consistent with its binding activity, BALB/c-derived macrophages exhibited a moderate inhibitory effect on human long-term culture-initiating cells in in vitro cultures, and showed moderate phagocytic activity against human hematopoietic stem cells. The increased affinity of BALB/c SIRPA for human CD47 was mounted at least through the BALB/c-specific L29V SNP within the IgV domain. Thus, the mouse strain effect on xenogeneic engraftment might be ascribed mainly to the binding affinity of strain-specific polymorphic SIRPA with human CD47. This information should be useful for developing a novel immunodeficient strain with superior efficiency for xenogeneic transplantation of human cells.

Crystallographic and NMR evidence for flexibility in oligosaccharyltransferases and its catalytic significance.

Oligosaccharyltransferase (OST) is a membrane-bound enzyme that catalyzes the transfer of an oligosaccharide to an asparagine residue in glycoproteins. It possesses a binding pocket that recognizes Ser and Thr residues at the +2 position in the N-glycosylation consensus, Asn-X-Ser/Thr. We determined the crystal structures of the C-terminal globular domains of the catalytic subunits of two archaeal OSTs. A comparison with previously determined structures identified a segment with remarkable conformational plasticity, induced by crystal contact effects. We characterized its dynamic properties in solution by (15)N NMR relaxation analyses. Intriguingly, the mobile region contains the +2 Ser/Thr-binding pocket. In agreement, the flexibility restriction forced by an engineered disulfide crosslink abolished the enzymatic activity, and its cleavage fully restored activity. These results suggest the necessity of multiple conformational states in the reaction. The dynamic nature of the Ser/Thr pocket could facilitate the efficient scanning of N-glycosylation sequons along nascent polypeptide chains.

Blockade of inflammatory responses by a small-molecule inhibitor of the Rac activator DOCK2.

Tissue infiltration of activated lymphocytes is a hallmark of transplant rejection and organ-specific autoimmune diseases. Migration and activation of lymphocytes depend on DOCK2, an atypical Rac activator predominantly expressed in hematopoietic cells. Although DOCK2 does not contain Dbl homology domain typically found in guanine nucleotide exchange factors, DOCK2 mediates the GTP-GDP exchange reaction for Rac through its DHR-2 domain. Here, we have identified 4-[3'-(2″-chlorophenyl)-2'-propen-1'-ylidene]-1-phenyl-3,5-pyrazolidinedione (CPYPP) as a small-molecule inhibitor of DOCK2. CPYPP bound to DOCK2 DHR-2 domain in a reversible manner and inhibited its catalytic activity in vitro. When lymphocytes were treated with CPYPP, both chemokine receptor- and antigen receptor-mediated Rac activation were blocked, resulting in marked reduction of chemotactic response and T cell activation. These results provide a rational of and a chemical scaffold for development of the DOCK2-targeting immunosuppressant.

Structural basis for mutual relief of the Rac guanine nucleotide exchange factor DOCK2 and its partner ELMO1 from their autoinhibited forms.

DOCK2, a hematopoietic cell-specific, atypical guanine nucleotide exchange factor, controls lymphocyte migration through ras-related C3 botulinum toxin substrate (Rac) activation. Dedicator of cytokinesis 2-engulfment and cell motility protein 1 (DOCK2•ELMO1) complex formation is required for DOCK2-mediated Rac signaling. In this study, we identified the N-terminal 177-residue fragment and the C-terminal 196-residue fragment of human DOCK2 and ELMO1, respectively, as the mutual binding regions, and solved the crystal structure of their complex at 2.1-Šresolution. The C-terminal Pro-rich tail of ELMO1 winds around the Src-homology 3 domain of DOCK2, and an intermolecular five-helix bundle is formed. Overall, the entire regions of both DOCK2 and ELMO1 assemble to create a rigid structure, which is required for the DOCK2•ELMO1 binding, as revealed by mutagenesis. Intriguingly, the DOCK2•ELMO1 interface hydrophobically buries a residue which, when mutated, reportedly relieves DOCK180 from autoinhibition. We demonstrated that the ELMO-interacting region and the DOCK-homology region 2 guanine nucleotide exchange factor domain of DOCK2 associate with each other for the autoinhibition, and that the assembly with ELMO1 weakens the interaction, relieving DOCK2 from the autoinhibition. The interactions between the N- and C-terminal regions of ELMO1 reportedly cause its autoinhibition, and binding with a DOCK protein relieves the autoinhibition for ras homolog gene family, member G binding and membrane localization. In fact, the DOCK2•ELMO1 interface also buries the ELMO1 residues required for the autoinhibition within the hydrophobic core of the helix bundle. Therefore, the present complex structure reveals the structural basis by which DOCK2 and ELMO1 mutually relieve their autoinhibition for the activation of Rac1 for lymphocyte chemotaxis.