Conformational Distribution of a Multidomain Protein Measured by Single-Pair Small-Angle X-ray Scattering.

The difficulty in evaluating the conformational distribution of proteins in solution often hinders mechanistic insights. One possible strategy for visualizing conformational distribution is distance distribution measurement by single-pair small-angle X-ray scattering (SAXS), in which the scattering interference from only a specific pair of atoms in the target molecule is extracted. Despite this promising concept, with few applications in synthetic small molecules and DNA, technical difficulties have prevented its application in protein conformational studies. This study used a synthetic tag to fix the lanthanide ion at desired sites on the protein and used single-pair SAXS with contrast matching to evaluate the conformational distribution of the multidomain protein enzyme MurD. These data highlighted the broad conformational and ligand-driven distribution shifts of MurD in solution. This study proposes an important strategy in solution structural biology that targets dynamic proteins, including multidomain and intrinsically disordered proteins.

Hydrolytic activity of yeast oligosaccharyltransferase is enhanced when misfolded proteins accumulate in the endoplasmic reticulum.

It is known that oligosaccharyltransferase (OST) has hydrolytic activity toward dolichol-linked oligosaccharides (DLO), which results in the formation of free N-glycans (FNGs), i.e. unconjugated oligosaccharides with structural features similar to N-glycans. The functional importance of this hydrolytic reaction, however, remains unknown. In this study, the hydrolytic activity of OST was characterized in yeast. It was shown that the hydrolytic activity of OST is enhanced in ubiquitin ligase mutants that are involved in endoplasmic reticulum-associated degradation. Interestingly, this enhanced hydrolysis activity is completely suppressed in asparagine-linked glycosylation (alg) mutants, bearing mutations related to the biosynthesis of DLO, indicating that the effect of ubiquitin ligase on OST-mediated hydrolysis is context-dependent. The enhanced hydrolysis activity in ubiquitin ligase mutants was also found to be canceled upon treatment of the cells with dithiothreitol, a reagent that potently induces protein unfolding in the endoplasmic reticulum (ER). Our results clearly suggest that the hydrolytic activity of OST is enhanced under conditions in which the formation of unfolded proteins is promoted in the ER in yeast. The possible role of FNGs on protein folding is discussed.

CHLOROPLAST UNUSUAL POSITIONING 1 is a plant-specific actin polymerization factor regulating chloroplast movement.

Plants have unique responses to fluctuating light conditions. One such response involves chloroplast photorelocation movement, which optimizes photosynthesis under weak light by the accumulation of chloroplasts along the periclinal side of the cell, which prevents photodamage under strong light by avoiding chloroplast positioning toward the anticlinal side of the cell. This light-responsive chloroplast movement relies on the reorganization of chloroplast actin (cp-actin) filaments. Previous studies have suggested that CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1) is essential for chloroplast photorelocation movement as a regulator of cp-actin filaments. In this study, we conducted comprehensive analyses to understand CHUP1 function. Functional, fluorescently tagged CHUP1 colocalized with and was coordinately reorganized with cp-actin filaments on the chloroplast outer envelope during chloroplast movement in Arabidopsis thaliana. CHUP1 distribution was reversibly regulated in a blue light- and phototropin-dependent manner. X-ray crystallography revealed that the CHUP1-C-terminal domain shares structural homology with the formin homology 2 (FH2) domain, despite lacking sequence similarity. Furthermore, the CHUP1-C-terminal domain promoted actin polymerization in the presence of profilin in vitro. Taken together, our findings indicate that CHUP1 is a plant-specific actin polymerization factor that has convergently evolved to assemble cp-actin filaments and enables chloroplast photorelocation movement.

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.

Effects of targeting signal mutations in a mitochondrial presequence on the spatial distribution of the conformational ensemble in the binding site of Tom20.

The 20-kDa TOM (translocase of outer mitochondrial membrane) subunit, Tom20, is the first receptor of the protein import pathway into mitochondria. Tom20 recognizes the mitochondrial targeting signal embedded in the presequences attached to mature mitochondrial proteins, as an N-terminal extension. Consequently, ~1,000 different mitochondrial proteins are sorted into the mitochondrial matrix, and distinguished from non-mitochondrial proteins. We previously reported the MPRIDE (multiple partial recognitions in dynamic equilibrium) mechanism to explain the structural basis of the promiscuous recognition of presequences by Tom20. A subset of the targeting signal features is recognized in each pose of the presequence in the binding state, and all of the features are collectively recognized in the dynamic equilibrium between the poses. Here, we changed the volumes of the hydrophobic side chains in the targeting signal, while maintaining the binding affinity. We tethered the mutated presequences to the binding site of Tom20 and placed them in the crystal contact-free space (CCFS) created in the crystal lattice. The spatial distributions of the mutated presequences were visualized as smeared electron densities in the low-pass filtered difference maps obtained by X-ray crystallography. The mutated presequence ensembles shifted their positions in the binding state to accommodate the larger side chains, thus providing positive evidence supporting the use of the MPRIDE mechanism in the promiscuous recognition by Tom20.

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.