Structural and Functional Analysis of the Amorphous Calcium Carbonate-Binding Protein Paramyosin in the Shell of the Pearl Oyster, Pinctada fucata.

Amorphous calcium carbonate (ACC) is an important precursor phase for the formation of aragonite crystals in the shells of Pinctada fucata. To identify the ACC-binding protein in the inner aragonite layer of the shell, extracts from the shell were used in the ACC-binding experiments. Semiquantitative analyses using liquid chromatography-mass spectrometry revealed that paramyosin was strongly associated with ACC in the shell. We discovered that paramyosin, a major component of the adductor muscle, was included in the myostracum, which is the microstructure of the shell attached to the adductor muscle. Purified paramyosin accumulates calcium carbonate and induces the prism structure of aragonite crystals, which is related to the morphology of prism aragonite crystals in the myostracum. Nuclear magnetic resonance measurements revealed that the Glu-rich region was bound to ACC. Activity of the Glu-rich region was stronger than that of the Asp-rich region. These results suggest that paramyosin in the adductor muscle is involved in the formation of aragonite prisms in the myostracum.

Molecular mechanism of glycolytic flux control intrinsic to human phosphoglycerate kinase.

Glycolysis plays a fundamental role in energy production and metabolic homeostasis. The intracellular [adenosine triphosphate]/[adenosine diphosphate] ([ATP]/[ADP]) ratio controls glycolytic flux; however, the regulatory mechanism underlying reactions catalyzed by individual glycolytic enzymes enabling flux adaptation remains incompletely understood. Phosphoglycerate kinase (PGK) catalyzes the reversible phosphotransfer reaction, which directly produces ATP in a near-equilibrium step of glycolysis. Despite extensive studies on the transcriptional regulation of PGK expression, the mechanism in response to changes in the [ATP]/[ADP] ratio remains obscure. Here, we report a protein-level regulation of human PGK (hPGK) by utilizing the switching ligand-binding cooperativities between adenine nucleotides and 3-phosphoglycerate (3PG). This was revealed by nuclear magnetic resonance (NMR) spectroscopy at physiological salt concentrations. MgADP and 3PG bind to hPGK with negative cooperativity, whereas MgAMPPNP (a nonhydrolyzable ATP analog) and 3PG bind to hPGK with positive cooperativity. These opposite cooperativities enable a shift between different ligand-bound states depending on the intracellular [ATP]/[ADP] ratio. Based on these findings, we present an atomic-scale description of the reaction scheme for hPGK under physiological conditions. Our results indicate that hPGK intrinsically modulates its function via ligand-binding cooperativities that are finely tuned to respond to changes in the [ATP]/[ADP] ratio. The alteration of ligand-binding cooperativities could be one of the self-regulatory mechanisms for enzymes in bidirectional pathways, which enables rapid adaptation to changes in the intracellular environment.

Structure and Dynamics of Drk-SH2 Domain and Its Site-Specific Interaction with Sev Receptor Tyrosine Kinase.

The Drosophila downstream receptor kinase (Drk), a homologue of human GRB2, participates in the signal transduction from the extracellular to the intracellular environment. Drk receives signals through the interaction of its Src homology 2 (SH2) domain with the phosphorylated tyrosine residue in the receptor tyrosine kinases (RTKs). Here, we present the solution NMR structure of the SH2 domain of Drk (Drk-SH2), which was determined in the presence of a phosphotyrosine (pY)-containing peptide derived from a receptor tyrosine kinase, Sevenless (Sev). The solution structure of Drk-SH2 possess a common SH2 domain architecture, consisting of three ß strands imposed between two α helices. Additionally, we interpret the site-specific interactions of the Drk-SH2 domain with the pY-containing peptide through NMR titration experiments. The dynamics of Drk-SH2 were also analysed through NMR-relaxation experiments as well as the molecular dynamic simulation. The docking simulations of the pY-containing peptide onto the protein surface of Drk-SH2 provided the orientation of the peptide, which showed a good agreement with the analysis of the SH2 domain of GRB2.

Interactions of the N- and C-Terminal SH3 Domains of Drosophila Drk with the Proline-Rich Peptides from Sos and Dos.

Drk, a homologue of human GRB2 in Drosophila, receives signals from outside the cells through the interaction of its SH2 domain with the phospho-tyrosine residues in the intracellular regions of receptor tyrosine kinases (RTKs) such as Sevenless, and transduces the signals downstream through the association of its N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) with proline-rich motifs (PRMs) in Son of Sevenless (Sos) or Daughter of Sevenless (Dos). Isolated Drk-NSH3 exhibits a conformational equilibrium between the folded and unfolded states, while Drk-CSH3 adopts only a folded confirmation. Drk interacts with PRMs of the PxxPxR motif in Sos and the PxxxRxxKP motif in Dos. Our previous study has shown that Drk-CSH3 can bind to Sos, but the interaction between Drk-NSH3 and Dos has not been investigated. To assess the affinities of both SH3 domains towards Sos and Dos, we conducted NMR titration experiments using peptides derived from Sos and Dos. Sos-S1 binds to Drk-NSH3 with the highest affinity, strongly suggesting that the Drk-Sos multivalent interaction is initiated by the binding of Sos-S1 and NSH3. Our results also revealed that the two Sos-derived PRMs clearly favour NSH3 for binding, whereas the two Dos-derived PRMs show almost similar affinity for NSH3 and CSH3. We have also performed docking simulations based on the chemical shift perturbations caused by the addition of Sos- and Dos-derived peptides. Finally, we discussed the various modes in the interactions of Drk with Sos/Dos.

Insight into the C-terminal SH3 domain mediated binding of Drosophila Drk to Sos and Dos.

Drk, a Drosophila homologue of human GRB2, interacts with Sevenless (Sev) receptor via its SH2 domain, while the N- and C-terminal SH3 domains (Drk-NSH3 and Drk-CSH3, respectively) are responsible for the interaction with proline-rich motifs (PRMs) of Son of sevenless (Sos) or Daughter of Sevenless (Dos). Drk-NSH3 on its own has a conformational equilibrium between folded and unfolded states, and the folded state is stabilised by the association with a Sos-derived proline-rich peptide with PxxPxR motif. In contrast, Drk-CSH3 is supposed to bind PxxxRxxKP motifs in Dos. Aiming at clarifying the structural and functional differences between the two SH3 domains, we performed NMR studies of Drk-CSH3. The resulting solution structure and the 15N-relaxation data showed that Drk-CSH3 consists of a stable domain. Large chemical shift perturbation was commonly found around the RT loop and the hydrophobic patch, while there were also changes that occur characteristically for Sos- or Dos-derived peptides. Sos-derived two peptides with PxxPxR motif showed stronger affinity to Drk-CSH3, indicating that the Sos PRMs can bind both N- and C-SH3 domains. Dos-derived two peptides could also bind Drk-CSH3, but with much weaker affinity, suggesting a possibility that any cooperative binding of Dos-PRMs may strengthen the Drk-Dos interaction. The NMR studies as well as the docking simulations provide valuable insights into the biological and biophysical functions of two SH3 domains in Drk.

Protein Structure Determination in Living Cells.

To date, in-cell NMR has elucidated various aspects of protein behaviour by associating structures in physiological conditions. Meanwhile, current studies of this method mostly have deduced protein states in cells exclusively based on 'indirect' structural information from peak patterns and chemical shift changes but not 'direct' data explicitly including interatomic distances and angles. To fully understand the functions and physical properties of proteins inside cells, it is indispensable to obtain explicit structural data or determine three-dimensional (3D) structures of proteins in cells. Whilst the short lifetime of cells in a sample tube, low sample concentrations, and massive background signals make it difficult to observe NMR signals from proteins inside cells, several methodological advances help to overcome the problems. Paramagnetic effects have an outstanding potential for in-cell structural analysis. The combination of a limited amount of experimental in-cell data with software for ab initio protein structure prediction opens an avenue to visualise 3D protein structures inside cells. Conventional nuclear Overhauser effect spectroscopy (NOESY)-based structure determination is advantageous to elucidate the conformations of side-chain atoms of proteins as well as global structures. In this article, we review current progress for the structure analysis of proteins in living systems and discuss the feasibility of its future works.

High-Resolution Protein 3D Structure Determination in Living Eukaryotic Cells.

Proteins in living cells interact specifically or nonspecifically with an enormous number of biomolecules. To understand the behavior of proteins under intracellular crowding conditions, it is indispensable to observe their three-dimensional (3D) structures at the atomic level in a physiologically natural environment. We demonstrate the first de novo protein structure determinations in eukaryotes with the sf9 cell/baculovirus system using NMR data from living cells exclusively. The method was applied to five proteins, rat calmodulin, human HRas, human ubiquitin, T. thermophilus HB8 TTHA1718, and Streptococcus protein G B1 domain. In all cases, we could obtain structural information from well-resolved in-cell 3D nuclear Overhauser effect spectroscopy (NOESY) data, suggesting that our method can be a standard tool for protein structure determinations in living eukaryotic cells. For three proteins, we achieved well-converged 3D structures. Among these, the in-cell structure of protein G B1 domain was most accurately determined, demonstrating that a helix-loop region is tilted away from a ß-sheet compared to the conformation in diluted solution.

Solution NMR views of dynamical ordering of biomacromolecules.

BACKGROUND: To understand the mechanisms related to the 'dynamical ordering' of macromolecules and biological systems, it is crucial to monitor, in detail, molecular interactions and their dynamics across multiple timescales. Solution nuclear magnetic resonance (NMR) spectroscopy is an ideal tool that can investigate biophysical events at the atomic level, in near-physiological buffer solutions, or even inside cells. SCOPE OF REVIEW: In the past several decades, progress in solution NMR has significantly contributed to the elucidation of three-dimensional structures, the understanding of conformational motions, and the underlying thermodynamic and kinetic properties of biomacromolecules. This review discusses recent methodological development of NMR, their applications and some of the remaining challenges. MAJOR CONCLUSIONS: Although a major drawback of NMR is its difficulty in studying the dynamical ordering of larger biomolecular systems, current technologies have achieved considerable success in the structural analysis of substantially large proteins and biomolecular complexes over 1MDa and have characterised a wide range of timescales across which biomolecular motion exists. While NMR is well suited to obtain local structure information in detail, it contributes valuable and unique information within hybrid approaches that combine complementary methodologies, including solution scattering and microscopic techniques. GENERAL SIGNIFICANCE: For living systems, the dynamic assembly and disassembly of macromolecular complexes is of utmost importance for cellular homeostasis and, if dysregulated, implied in human disease. It is thus instructive for the advancement of the study of the dynamical ordering to discuss the potential possibilities of solution NMR spectroscopy and its applications. This article is part of a Special Issue entitled "Biophysical Exploration of Dynamical Ordering of Biomolecular Systems" edited by Dr. Koichi Kato.

A new carbamidemethyl-linked lanthanoid chelating tag for PCS NMR spectroscopy of proteins in living HeLa cells.

Structural analyses of proteins under macromolecular crowding inside human cultured cells by in-cell NMR spectroscopy are crucial not only for explicit understanding of their cellular functions but also for applications in medical and pharmaceutical sciences. In-cell NMR experiments using human cultured cells however suffer from low sensitivity, thus pseudocontact shifts from protein-tagged paramagnetic lanthanoid ions, analysed using sensitive heteronuclear two-dimensional correlation NMR spectra, offer huge potential advantage in obtaining structural information over conventional NOE-based approaches. We synthesised a new lanthanoid-chelating tag (M8-CAM-I), in which the eight-fold, stereospecifically methylated DOTA (M8) scaffold was retained, while a stable carbamidemethyl (CAM) group was introduced as the functional group connecting to proteins. M8-CAM-I successfully fulfilled the requirements for in-cell NMR: high-affinity to lanthanoid, low cytotoxicity and the stability under reducing condition inside cells. Large PCSs for backbone N-H resonances observed for M8-CAM-tagged human ubiquitin mutant proteins, which were introduced into HeLa cells by electroporation, demonstrated that this approach readily provides the useful information enabling the determination of protein structures, relative orientations of domains and protein complexes within human cultured cells.

Evaluation of the reliability of the maximum entropy method for reconstructing 3D and 4D NOESY-type NMR spectra of proteins.

Despite their advantages in analysis, 4D NMR experiments are still infrequently used as a routine tool in protein NMR projects due to the long duration of the measurement and limited digital resolution. Recently, new acquisition techniques for speeding up multidimensional NMR experiments, such as nonlinear sampling, in combination with non-Fourier transform data processing methods have been proposed to be beneficial for 4D NMR experiments. Maximum entropy (MaxEnt) methods have been utilised for reconstructing nonlinearly sampled multi-dimensional NMR data. However, the artefacts arising from MaxEnt processing, particularly, in NOESY spectra have not yet been clearly assessed in comparison with other methods, such as quantitative maximum entropy, multidimensional decomposition, and compressed sensing. We compared MaxEnt with other methods in reconstructing 3D NOESY data acquired with variously reduced sparse sampling schedules and found that MaxEnt is robust, quick and competitive with other methods. Next, nonlinear sampling and MaxEnt processing were applied to 4D NOESY experiments, and the effect of the artefacts of MaxEnt was evaluated by calculating 3D structures from the NOE-derived distance restraints. Our results demonstrated that sufficiently converged and accurate structures (RMSD of 0.91Å to the mean and 1.36Å to the reference structures) were obtained even with NOESY spectra reconstructed from 1.6% randomly selected sampling points for indirect dimensions. This suggests that 3D MaxEnt processing in combination with nonlinear sampling schedules is still a useful and advantageous option for rapid acquisition of high-resolution 4D NOESY spectra of proteins.

Protein structure determination in living cells by in-cell NMR spectroscopy.

Investigating proteins 'at work' in a living environment at atomic resolution is a major goal of molecular biology, which has not been achieved even though methods for the three-dimensional (3D) structure determination of purified proteins in single crystals or in solution are widely used. Recent developments in NMR hardware and methodology have enabled the measurement of high-resolution heteronuclear multi-dimensional NMR spectra of macromolecules in living cells (in-cell NMR). Various intracellular events such as conformational changes, dynamics and binding events have been investigated by this method. However, the low sensitivity and the short lifetime of the samples have so far prevented the acquisition of sufficient structural information to determine protein structures by in-cell NMR. Here we show the first, to our knowledge, 3D protein structure calculated exclusively on the basis of information obtained in living cells. The structure of the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB8 overexpressed in Escherichia coli cells was solved by in-cell NMR. Rapid measurement of the 3D NMR spectra by nonlinear sampling of the indirectly acquired dimensions was used to overcome problems caused by the instability and low sensitivity of living E. coli samples. Almost all of the expected backbone NMR resonances and most of the side-chain NMR resonances were observed and assigned, enabling high quality (0.96 ångström backbone root mean squared deviation) structures to be calculated that are very similar to the in vitro structure of TTHA1718 determined independently. The in-cell NMR approach can thus provide accurate high-resolution structures of proteins in living environments.

Structural insights into the complex of oncogenic KRas4BG12V and Rgl2, a RalA/B activator.

About a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA, and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human KRas4B and the Ras association (RA) domain of Rgl2 (Rgl2RA), one of the RA-containing RalGEFs. We show that the G12V oncogenic KRas4B mutation changes the interaction kinetics with Rgl2RA The crystal structure of the KRas4BG12V: Rgl2RA complex shows a 2:2 heterotetramer where the switch I and switch II regions of each KRasG12V interact with both Rgl2RA molecules. This structural arrangement is highly similar to the HRasE31K:RALGDSRA crystal structure and is distinct from the well-characterised Ras:Raf complex. Interestingly, the G12V mutation was found at the dimer interface of KRas4BG12V with its partner. Our study reveals a potentially distinct mode of Ras:effector complex formation by RalGEFs and offers a possible mechanistic explanation for how the oncogenic KRas4BG12V hyperactivates the RalA/B pathway.

High-resolution heteronuclear multidimensional NMR of proteins in living insect cells using a baculovirus protein expression system.

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. In order to complement the existing protocols, and to extend the range of possible applications, we introduce a novel approach for observing in-cell NMR spectra using the sf9 cell/baculovirus system. High-resolution 2D (1)H-(15)N correlation spectra were observed for four model proteins expressed in sf9 cells. Furthermore, 3D triple-resonance NMR spectra of the Streptococcus protein G B1 domain were observed in sf9 cells by using nonlinear sampling to overcome the short lifetime of the samples and the low abundance of the labeled protein. The data were processed with a quantitative maximum entropy algorithm. These were assigned ab initio, yielding approximately 80% of the expected backbone NMR resonances. Well-resolved NOE cross peaks could be identified in the 3D (15)N-separated NOESY spectrum, suggesting that structural analysis of this size of protein will be feasible in sf9 cells.

An in-cell NMR study of monitoring stress-induced increase of cytosolic Ca2+ concentration in HeLa cells.

Recent developments in in-cell NMR techniques have allowed us to study proteins in detail inside living eukaryotic cells. The lifetime of in-cell NMR samples is however much shorter than that in culture media, presumably because of various stresses as well as the nutrient depletion in the anaerobic environment within the NMR tube. It is well known that Ca(2+)-bursts occur in HeLa cells under various stresses, hence the cytosolic Ca(2+) concentration can be regarded as a good indicator of the healthiness of cells in NMR tubes. In this study, aiming at monitoring the states of proteins resulting from the change of cytosolic Ca(2+) concentration during experiments, human calbindin D9k (P47M+C80) was used as the model protein and cultured HeLa cells as host cells. Time-resolved measurements of 2D (1)H-(15)N SOFAST-HMQC experiments of calbindin D9k (P47M+C80) in HeLa cells showed time-dependent changes in the cross-peak patterns in the spectra. Comparison with in vitro assignments revealed that calbindin D9k (P47M+C80) is initially in the Mg(2+)-bound state, and then gradually converted to the Ca(2+)-bound state. This conversion process initiates after NMR sample preparation. These results showed, for the first time, that cells inside the NMR tube were stressed, presumably because of cell precipitation, the lack of oxygen and nutrients, etc., thereby releasing Ca(2+) into cytosol during the measurements. The results demonstrated that in-cell NMR can monitor the state transitions of stimulated cells through the observation of proteins involved in the intracellular signalling systems. Our method provides a very useful tool for in situ monitoring of the "healthiness" of the cells in various in-cell NMR studies.

Structural investigation of the C-terminal catalytic fragment of presenilin 1.

The gamma-secretase complex has a decisive role in the development of Alzheimer's disease, in that it cleaves a precursor to create the amyloid beta peptide whose aggregates form the senile plaques encountered in the brains of patients. Gamma-secretase is a member of the intramembrane-cleaving proteases which process their transmembrane substrates within the bilayer. Many of the mutations encountered in early onset familial Alzheimer's disease are linked to presenilin 1, the catalytic component of gamma-secretase, whose active form requires its endoproteolytic cleavage into N-terminal and C-terminal fragments. Although there is general agreement regarding the topology of the N-terminal fragment, studies of the C-terminal fragment have yielded ambiguous and contradictory results that may be difficult to reconcile in the absence of structural information. Here we present the first structure of the C-terminal fragment of human presenilin 1, as obtained from NMR studies in SDS micelles. The structure reveals a topology where the membrane is likely traversed three times in accordance with the more generally accepted nine transmembrane domain model of presenilin 1, but contains unique structural features adapted to accommodate the unusual intramembrane catalysis. These include a putative half-membrane-spanning helix N-terminally harboring the catalytic aspartate, a severely kinked helical structure toward the C terminus as well as a soluble helix in the assumed-to-be unstructured N-terminal loop.

NMR solution structure of a Chymotrypsin inhibitor from the Taiwan cobra Naja naja atra.

The Taiwan cobra (Naja naja atra) chymotrypsin inhibitor (NACI) consists of 57 amino acids and is related to other Kunitz-type inhibitors such as bovine pancreatic trypsin inhibitor (BPTI) and Bungarus fasciatus fraction IX (BF9), another chymotrypsin inhibitor. Here we present the solution structure of NACI. We determined the NMR structure of NACI with a root-mean-square deviation of 0.37 Å for the backbone atoms and 0.73 Å for the heavy atoms on the basis of 1,075 upper distance limits derived from NOE peaks measured in its NOESY spectra. To investigate the structural characteristics of NACI, we compared the three-dimensional structure of NACI with BPTI and BF9. The structure of the NACI protein comprises one 310-helix, one α-helix and one double-stranded antiparallel ß-sheet, which is comparable with the secondary structures in BPTI and BF9. The RMSD value between the mean structures is 1.09 Å between NACI and BPTI and 1.27 Å between NACI and BF9. In addition to similar secondary and tertiary structure, NACI might possess similar types of protein conformational fluctuations as reported in BPTI, such as Cys14-Cys38 disulfide bond isomerization, based on line broadening of resonances from residues which are mainly confined to a region around the Cys14-Cys38 disulfide bond.

Exclusively NOESY-based automated NMR assignment and structure determination of proteins.

A fully automated method is presented for determining NMR solution structures of proteins using exclusively NOESY spectra as input, obviating the need to measure any spectra only for obtaining resonance assignments but devoid of structural information. Applied to two small proteins, the approach yielded structures that coincided closely with conventionally determined structures.

Targeting chromosome trisomy for chromosome editing.

A trisomy is a type of aneuploidy characterised by an additional chromosome. The additional chromosome theoretically accepts any kind of changes since it is not necessary for cellular proliferation. This advantage led us to apply two chromosome manipulation methods to autosomal trisomy in chicken DT40 cells. We first corrected chromosome 2 trisomy to disomy by employing counter-selection markers. Upon construction of cells carrying markers targeted in one of the trisomic chromosome 2s, cells that have lost markers integrated in chromosome 2 were subsequently selected. The loss of one of the chromosome 2s had little impacts on the proliferative capacity, indicating unsubstantial role of the additional chromosome 2 in DT40 cells. We next tested large-scale truncations of chromosome 2 to make a mini-chromosome for the assessment of chromosome stability by introducing telomere repeat sequences to delete most of p-arm or q-arm of chromosome 2. The obtained cell lines had 0.7 Mb mini-chromosome, and approximately 0.2% of mini-chromosome was lost per cell division in wild-type background while the rate of chromosome loss was significantly increased by the depletion of DDX11, a cohesin regulatory protein. Collectively, our findings propose that trisomic chromosomes are good targets to make unique artificial chromosomes.

Automated NMR structure determination of stereo-array isotope labeled ubiquitin from minimal sets of spectra using the SAIL-FLYA system.

Stereo-array isotope labeling (SAIL) has been combined with the fully automated NMR structure determination algorithm FLYA to determine the three-dimensional structure of the protein ubiquitin from different sets of input NMR spectra. SAIL provides a complete stereo- and regio-specific pattern of stable isotopes that results in sharper resonance lines and reduced signal overlap, without information loss. Here we show that as a result of the superior quality of the SAIL NMR spectra, reliable, fully automated analyses of the NMR spectra and structure calculations are possible using fewer input spectra than with conventional uniformly 13C/15N-labeled proteins. FLYA calculations with SAIL ubiquitin, using a single three-dimensional "through-bond" spectrum (and 2D HSQC spectra) in addition to the 13C-edited and 15N-edited NOESY spectra for conformational restraints, yielded structures with an accuracy of 0.83-1.15 A for the backbone RMSD to the conventionally determined solution structure of SAIL ubiquitin. NMR structures can thus be determined almost exclusively from the NOESY spectra that yield the conformational restraints, without the need to record many spectra only for determining intermediate, auxiliary data of the chemical shift assignments. The FLYA calculations for this report resulted in 252 ubiquitin structure bundles, obtained with different input data but identical structure calculation and refinement methods. These structures cover the entire range from highly accurate structures to seriously, but not trivially, wrong structures, and thus constitute a valuable database for the substantiation of structure validation methods.

Paramagnetic relaxation enhancement-assisted structural characterization of a partially disordered conformation of ubiquitin.

Nuclear magnetic resonance (NMR) is a powerful tool to study three-dimensional structures as well as protein conformational fluctuations in solution, but it is compromised by increases in peak widths and missing signals. We previously reported that ubiquitin has two folded conformations, N1 and N2 and plus another folded conformation, I, in which some amide group signals of residues 33-41 almost disappeared above 3 kbar at pH 4.5 and 273 K. Thus, well-converged structural models could not be obtained for this region owing to the absence of distance restraints. Here, we reexamine the problem using the ubiquitin Q41N variant as a model for this locally disordered conformation, I. We demonstrate that the variant shows pressure-induced loss of backbone amide group signals at residues 28, 33, 36, and 39-41 like the wild-type, with a similar but smaller effect on CαH and CßH signals. In order to characterize this I structure, we measured paramagnetic relaxation enhancement (PRE) under high pressure to obtain distance restraints, and calculated the structure assisted by Bayesian inference. We conclude that the more disordered I conformation observed at pH 4.0, 278 K, and 2.5 kbar largely retained the N2 conformation, although the amide groups at residues 33-41 have more heterogeneous conformations and more contact with water, which differ from the N1 and N2 states. The PRE-assisted strategy has the potential to improve structural characterization of proteins that lack NMR signals, especially for relatively more open and hydrated protein conformations.