Relaxation Dispersion Experiments Research Articles

Structural dynamics of human deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase)

Structural- and functional heterogeneity, as well as allosteric regulation, in homo-monomeric enzymes is a highly active area of research. One such enzyme is human nuclear-associated deoxyuridine 5’-triphosphate nucleotidohydrolase (dUTPase), which has emerged as an interesting drug target in combination therapy with traditional nucleotide analogue treatment of cancer. We report, for the first time, a full structural dynamics study of human dUTPase by NMR. dUTPase has been investigated in terms of structural dynamics in its apo form, in complex with the modified substrate resistant to hydrolysis, 2’-deoxyuridine 5’-α,β-imido-triphosphate (dUpNHpp), as well as the product, 2’-deoxy-uridine-monophosphate (dUMP). The apo form of the enzyme displayed slow dynamics in the milli- to microsecond regime in relaxation dispersion experiments, which was further slowed down to observable heterogeneity upon substrate-analogue binding. The results suggest that the non-hydrolysable substrate-analogue traps the enzyme in the conformational isomerization step that has been previously suggested to be part of the enzyme catalysis kinetics cycle. The observed heterogeneity fits well with the pattern expected to emerge from the suggested kinetic model, and no evidence for homotropic allosterism was found. The heatmaps of the slow dynamics, chemical shift perturbation upon substrate binding and conserved regions of the enzyme sequence all displayed a similar pattern, which suggests that the structural dynamics is finely tuned and important for the biological function of the enzyme for binding, conformational shift, catalysis and substrate release.

Insights into Ligand-Mediated Activation of an Oligomeric Ring-Shaped Gene-Regulatory Protein from Solution- and Solid-State NMR

The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13C–13C and 15N-13C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the µs-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity.

Existence of Singularities in NMR Relaxation Dispersion Profiles: Implications for Hidden Dynamics.

It is common for NMR relaxation dispersion experiments to suggest an absence of dynamics despite anecdotal indications of conformational dynamics. We explore the potential explanations and approaches to this conundrum. Some inconsistencies have been observed between two relaxation dispersion experiments, Carr-Purcell-Meiboom-Gill (CPMG) and adiabatic relaxation dispersion experiments, in recent dynamic studies of different biomolecules. Theoretical analyses show that such seemingly paradoxical results might come from a complex exchange topology that is concealed by the application of the simple two-site exchange model for interpretation of the relaxation dispersion data. Scenarios are explored and revealed in which the presence of complex millisecond conformational exchange could suppress the amplitude of CPMG relaxation dispersion profiles, even when the exchange rates are within the detectable range of the experiments. With experimental errors, the suppressed relaxation dispersion profiles could lead to the conclusion of "no millisecond conformational exchange". However, such hidden dynamics can potentially be detected by adiabatic relaxation dispersion experiments. Finally, we demonstrate some advantages of adiabatic relaxation dispersion experiments over conventional relaxation dispersion experiments and a simplified computational approach to analyze the adiabatic relaxation dispersion profiles.

From Milliseconds to Minutes: Melittin Self-Assembly from Concerted Non-Equilibrium Pressure-Jump and Equilibrium Relaxation Nuclear Magnetic Resonance.

Non-equilibrium kinetics techniques like pressure-jump nuclear magnetic resonance (NMR) are powerful in tracking changes in oligomeric populations and are not limited by relaxation rates for the time scales of exchange that can be probed. However, these techniques are less sensitive to minor, transient populations than are Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion experiments. We integrated non-equilibrium pressure-jump and equilibrium CPMG relaxation dispersion data to fully map the kinetic landscape of melittin tetramerization. While monomeric peptides weakly form dimers (Kd,D/M ≈ 26 mM) whose population never exceeds 1.6% at 288 K, dimers associate tightly to form stable tetrameric species (Kd,T/D ≈ 740 nM). Exchange between the monomer and dimer, along with exchange between the dimer and tetramer, occurs on the millisecond time scale. The NMR approach developed herein can be readily applied to studying the folding and misfolding of a wide range of oligomeric assemblies.

Optimization of TROSY- and anti-TROSY-based 15N CPMG relaxation dispersion experiments through phase cycling

CPMG relaxation dispersion studies of biomolecular dynamics on the μs–ms timescale can provide detailed kinetic, thermodynamic, and structural insights into function. Frequently, the 15N spin serves as the probe of choice, as uniform incorporation of the 15N isotope is facile and cost-effective, and the interpretation of the resulting data is often relatively straightforward. In conventional CPMG relaxation dispersion experiments the application of CPMG pulses with constant radiofrequency (RF) phase can lead to artifactual dispersion profiles that result from off-resonance effects, RF field inhomogeneity, and pulse miscalibration. The development of CPMG experiments with the [0013]-phase cycle has significantly reduced the impact of pulse imperfections over a greater bandwidth of frequency offsets in comparison to constant phase experiments. Application of 15N-TROSY-based CPMG schemes to studies of the dynamics of large molecules is necessary for high sensitivity, yet the correct incorporation of the [0013]-phase cycle is non-trivial. Here we present TROSY- and anti-TROSY-based 15N CPMG experiments with the [0013]-phase cycling scheme and demonstrate, through comprehensive numerical simulations and experimental validation, enhanced resistance to pulse imperfections relative to traditional schemes utilizing constant phase CPMG pulses. Notably, exchange parameters derived from the new experiments are in good agreement with those obtained using other, more established, 15N-based CPMG approaches.

Metal Binding of Alzheimer's Amyloid-β and Its Effect on Peptide Self-Assembly.

ConspectusMetal ions have been identified as key factors modulating the aggregation of amyloid-β peptide (Aβ) implicated in Alzheimer's disease (AD). The presence of elevated levels of metal ions in the amyloid plaques in AD patients supports the notion that the dysfunction of metal homeostasis is connected to the development of AD pathology. Here, recent findings from high- and low-resolution biophysical techniques are put into perspective, providing detailed insights into the molecular structures and dynamics of metal-bound Aβ complexes and the effect of metal ions on the Aβ aggregation process. In particular, the development of theoretical kinetic models deducing different microscopic nucleation events from the macroscopic aggregation behavior has enabled deciphering of the effect of metal ions on specific nucleation processes. In addition to these macroscopic measurements of bulk aggregation to quantify microscopic rates, recent NMR studies have revealed details about the structures and dynamics of metal-Aβ complexes, thereby linking structural events to bulk aggregation. Interestingly, transition-metal ions, such as copper, zinc, and silver ions, form a compact complex with the N-terminal part of monomeric Aβ, respectively, where the metal-bound "folded" state is in dynamic equilibrium with an "unfolded" state. The rates and thermodynamic features of these exchange dynamics have been determined by using NMR relaxation dispersion experiments. Additionally, the application of specifically tailored paramagnetic NMR experiments on the Cu(II)-Aβ complex has been fruitful in obtaining structural constraints within the blind sphere of conventional NMR experiments. This enables the determination of molecular structures of the "folded" Cu(II)-coordinated N-terminal region of Aβ. Furthermore, the discussed transition-metal ions modulate Aβ self-assembly in a concentration-dependent manner, where low metal ion concentrations inhibit Aβ fibril formation, while at high metal ion concentrations other processes occur, resulting in amorphous aggregate formation. Remarkably, the metal-Aβ interaction predominately reduces one specific nucleation step, the fibril-end elongation, whereas primary and surface-catalyzed secondary nucleation mechanisms are less affected. Specific inhibition of fibril-end elongation theoretically predicts an enhanced generation of Aβ oligomers, which is an interesting contribution to understanding metal-Aβ-associated neurotoxic effects. Taken together, the metal binding process creates a metal-bound Aβ complex, which is seemingly inert to aggregation. This process hence efficiently reduces the aggregation-prone peptide pool, which on the macroscopic level is reflected as slower aggregation kinetics. Thus, the specific binding of metals to the Aβ monomer can be linked to the macroscopic inhibitory effect on Aβ bulk aggregation, providing a molecular understanding of the Aβ aggregation mechanism in the presence of metal ions, where the metal ion can be seen as a minimalist agent against Aβ self-assembly. These insights can help to target Aβ aggregation in vivo, where metal ions are key factors modulating the Aβ self-assembly and Aβ-associated neurotoxicity.

An integrated approach of NMR experiments and MD simulations visualizes structural dynamics of a cyclic multi-domain protein.

Cyclization can stabilize the structure of proteins, as previously demonstrated in single-domain proteins. Although Lys48-linked polyubiquitin, a multi-domain protein, is also known to be cyclized in human cells, the structural effects of cyclization remain unclear. Here, we examined the impact of cyclization on the structural stability and dynamics of cyclic Lys48-linked diubiquitin (Ub2 ). As expected, cyclization increased the thermal stability of Ub2 and its resistance to proteolytic digestion, indicating that cyclization stabilized the structure of Ub2 . Furthermore, cyclization repressed the interdomain motion in Ub2 , but cyclic Ub2 still exhibited microsecond conformational exchange in NMR relaxation dispersion experiments. A series of long coarse-grained (CG) MD simulations visualized how cyclization slowed down the intrinsic nanosecond open-closed domain motion of Ub2 to microseconds. Thus, CG-MD analysis helped to explain the unexpected NMR relaxation results, thereby facilitating characterization of the structural stabilization of cyclic Ub2 .

Excited-state observation of active K-Ras reveals differential structural dynamics of wild-type versus oncogenic G12D and G12C mutants

Despite the prominent role of the K-Ras protein in many different types of human cancer, major gaps in atomic-level information severely limit our understanding of its functions in health and disease. Here, we report the quantitative backbone structural dynamics of K-Ras by solution nuclear magnetic resonance spectroscopy of the active state of wild-type K-Ras bound to guanosine triphosphate (GTP) nucleotide and two of its oncogenic P-loop mutants, G12D and G12C, using a new nanoparticle-assisted spin relaxation method, relaxation dispersion and chemical exchange saturation transfer experiments covering the entire range of timescales from picoseconds to milliseconds. Our combined experiments allow detection and analysis of the functionally critical Switch I and Switch II regions, which have previously remained largely unobservable by X-ray crystallography and nuclear magnetic resonance spectroscopy. Our data reveal cooperative transitions of K-Ras·GTP to a highly dynamic excited state that closely resembles the partially disordered K-Ras·GDP state. These results advance our understanding of differential GTPase activities and signaling properties of the wild type versus mutants and may thus guide new strategies for the development of therapeutics.

Reduced dynamic complexity allows structure elucidation of an excited state of KRASG13D

Localized dynamics of RAS, including regions distal to the nucleotide-binding site, is of high interest for elucidating the mechanisms by which RAS proteins interact with effectors and regulators and for designing inhibitors. Among several oncogenic mutants, methyl relaxation dispersion experiments reveal highly synchronized conformational dynamics in the active (GMPPNP-bound) KRASG13D, which suggests an exchange between two conformational states in solution. Methyl and 31P NMR spectra of active KRASG13D in solution confirm a two-state ensemble interconverting on the millisecond timescale, with a major Pγ atom peak corresponding to the dominant State 1 conformation and a secondary peak indicating an intermediate state different from the known State 2 conformation recognized by RAS effectors. High-resolution crystal structures of active KRASG13D and KRASG13D-RAF1 RBD complex provide snapshots of the State 1 and 2 conformations, respectively. We use residual dipolar couplings to solve and cross-validate the structure of the intermediate state of active KRASG13D, showing a conformation distinct from those of States 1 and 2 outside the known flexible switch regions. The dynamic coupling between the conformational exchange in the effector lobe and the breathing motion in the allosteric lobe is further validated by a secondary mutation in the allosteric lobe, which affects the conformational population equilibrium.

Insights into the Structure of Invisible Conformations of Large Methyl Group Labeled Molecular Machines from High Pressure NMR

Most proteins are highly flexible and can adopt conformations that deviate from the energetically most favorable ground state. Structural information on these lowly populated, alternative conformations is often lacking, despite the functional importance of these states. Here, we study the pathway by which the Dcp1:Dcp2 mRNA decapping complex exchanges between an autoinhibited closed and an open conformation. We make use of methyl Carr–Purcell–Meiboom–Gill (CPMG) NMR relaxation dispersion (RD) experiments that report on the population of the sparsely populated open conformation as well as on the exchange rate between the two conformations. To obtain volumetric information on the open conformation as well as on the transition state structure we made use of RD measurements at elevated pressures. We found that the open Dcp1:Dcp2 conformation has a lower molecular volume than the closed conformation and that the transition state is close in volume to the closed state. In the presence of ATP the volume change upon opening of the complex increases and the volume of the transition state lies in-between the volumes of the closed and open state. These findings show that ATP has an effect on the volume changes that are associated with the opening-closing pathway of the complex. Our results highlight the strength of pressure dependent NMR methods to obtain insights into structural features of protein conformations that are not directly observable. As our work makes use of methyl groups as NMR probes we conclude that the applied methodology is also applicable to high molecular weight complexes.

Slow conformational changes in the rigid and highly stable chymotrypsin inhibitor 2.

Slow conformational changes are often directly linked to protein function. It is however less clear how such processes may perturb the overall folding stability of a protein. We previously found that the stabilizing double mutant L49I/I57V in the small protein chymotrypsin inhibitor 2 from barley led to distributed increased nanosecond and faster dynamics. Here we asked what effects the L49I and I57V substitutions, either individually or together, have on the slow conformational dynamics of CI2. We used 15 N CPMG spin relaxation dispersion experiments to measure the kinetics, thermodynamics and structural changes associated with slow conformational change in CI2. These changes result in an excited state that is populated to 4.3% at 1 °C. As the temperature is increased the population of the excited state decreases. Structural changes in the excited state are associated with residues that interact with water molecules that have well defined positions and are found at these positions in all crystal structures of CI2. The substitutions in CI2 have only little effect on the structure of the excited state whereas the stability of the excited state to some extent follows the stability of the main state. The minor state is thus most populated for the most stable CI2 variant and least populated for the least stable variant. We hypothesize that the interactions between the substituted residues and the well-ordered water molecules links subtle structural changes around the substituted residues to the region in the protein that experience slow conformational changes. This article is protected by copyright. All rights reserved.

Site-based description of R1ρ relaxation in local reference frames

Nuclear magnetic spin relaxation in the presence of an applied radiofrequency field depends critically on chemical exchange processes that transfer nuclear spins between chemical or conformational environments with distinct resonance frequencies. Characterization of chemical exchange processes in R1ρ relaxation dispersion, CEST, and DEST experiments provides powerful insights into chemical and conformational kinetics of biological macromolecules. The present work reformulates expressions for magnetization evolution and the R1ρ relaxation rate constant by focussing on the orientations of the tilted rotating frames of reference for magnetization components in different sites, by treating the spin-locking field strength as a perturbation to free-precession evolution, and by applying the Homotopy Analysis and Laplace transform methods to approximate solutions to the Bloch-McConnell equations. The results provide an expression for R1ρ that is invariant to the topology of the kinetic scheme, an approximate equation for evolution of spin-locked z-magnetization, and an approach for effective simplification of chemical exchange topologies for 2- and N-site chemical exchange processes. The theoretical approach also provides an accurate approximation for relaxation during a constant-amplitude radiofrequency field in the absence of exchange.

SPICY: a method for single scan rotating frame relaxometry.

T 1ρ is an NMR relaxation mode that is sensitive to low frequency molecular motions, making it an especially valuable tool in biomolecular research. Here, we introduce a new method, SPICY, for measuring T1ρ relaxation times. In contrast to conventional T1ρ experiments, in which the sequence is repeated many times to determine the T1ρ time, the SPICY sequence allows determination of T1ρ within a single scan, shortening the experiment time remarkably. We demonstrate the method using 1H T1ρ relaxation dispersion experiments. Additionally, we combine the sequence with spatial encoding to produce 1D images in a single scan. We show that T1ρ relaxation times obtained using the single scan approach are in good agreement with those obtained using the traditional experiments.

Transient On- and Off-Pathway Protein Folding Intermediate States Characterized with NMR Relaxation Dispersion.

The earliest events in the folding of a protein are in general poorly understood. We used NMR R2 relaxation dispersion experiments to study transient local collapse events in the unfolded-state (U) conformational ensemble of apomyoglobin (apoMb). Local residual secondary structure (seen in regions corresponding to the A, D, E, and H helices of the folded protein) is largely unchanged over the pH range of 2.3-2.75, yet a significant pH-dependent increase in the conformational exchange contribution to the R2 relaxation rate (Rex) indicates that transient intramolecular contacts occur on a microsecond to millisecond time scale at pH 2.75. A comparison of 15N and 13CO relaxation dispersion data at pH 2.75 for residues in the A, B, G, and H regions, which participate in the earliest folding intermediates, indicates that chain collapse and secondary structure formation are rapid and concomitant. Increasingly stabilizing conditions (lower temperature, higher pH) result in the observation of a relaxation dispersion in the C, CD, and E regions of the protein, which are known to fold at later stages. Mutation of Trp14 in the A-helix region to Ala eliminates conformational exchange throughout the protein, and the mutation of hydrophobic residues in other regions results in the selective inhibition of conformational exchange in the B, G, or H regions. The R2 dispersion data for WT apoMb at pH 2.75 and 10 °C are best fit to a four-state model ABGH ⇆ AGH ⇆ U ⇆ ABCD that includes on-pathway (AGH and ABGH) and off-pathway (ABCD) transiently folded states, both of which are required to explain the behavior of the mutant proteins. The off-pathway intermediate is destabilized at higher temperatures. Our analysis provides insights into the earliest stages of apoMb folding where the collapsing polypeptide chain samples both productive and nonproductive states with stabilized secondary structure.

Evolutionary Role of Water-Accessible Cavities in Src Homology 2 (SH2) Domains.

Protein excited states are fundamental in the understanding of biological function, despite the fact they are hardly observed using traditional biophysical methodologies. Pressure perturbation coupled with nuclear magnetic resonance (NMR) spectroscopy is a powerful physicochemical tool to glance at these low-populated high-energy states on a residue-by-residue basis and underpin mechanistic insights into protein functionalities. Here we performed pressure titrations using NMR spectroscopy and relaxation dispersion experiments to identify the low-lying energetic states of the c-Abl SH2 domain. By showing that the SH2 excited state contains a hydrated hydrophobic cavity, fast-exchange motions, and highly conserved residues facing the water-accessible hole, we discuss the implications of water-protein interactions in SH2 modules achieving high-affinity binding and promiscuous phospho-Tyr peptide recognition.

Characterizing Fast Conformational Exchange of Aromatic Rings Using Residual Dipolar Couplings: Distinguishing Jumplike Flips from Other Exchange Mechanisms.

Aromatic ring flips are a hallmark of protein dynamics. They are experimentally studied by NMR spectroscopy, where recent advances have led to improved characterization across a wide range of time scales. Results on different proteins have been interpreted as continuous diffusive ring rotations or jumplike flips, leading to diverging views of the protein interior as being fluidlike or solidlike, respectively. It is challenging to distinguish between these mechanisms and other types of conformational exchange because chemical-shift-mediated line broadening provides only conclusive evidence for ring flips only if the system can be moved from the slow- to intermediate/fast-exchange regime. Moreover, whenever the chemical shift difference between the two symmetry-related sites is close to zero, it is not generally possible to determine the exchange time scale. Here we resolve these issues by measuring residual dipolar coupling (RDC)-mediated exchange contributions using NMR relaxation dispersion experiments on proteins dissolved in dilute liquid crystalline media. Excellent agreement is found between the experimental difference in RDC between the two symmetry-related sites and the value calculated from high-resolution X-ray structures, demonstrating that dynamics measured for F52 in the B1 domain of protein G reports on distinct, jumplike flips rather than other types of conformational exchange.

Molecular Insights into the Intrinsic Dynamics and Their Roles During Catalysis in Pin1 Peptidyl-prolyl Isomerase.

Proteins are intrinsically dynamic and change conformations over a wide range of time scales. While the conformational dynamics have been realized to be important for protein functions, e.g., in activity-stability trade-offs, how they play a role during enzyme catalysis has been of debate over decades. By studying Pin1 peptidyl-prolyl isomerase using extensive molecular dynamics simulations, here we discuss how the slow intrinsic dynamics of Pin1 observed in the NMR relaxation dispersion experiment occur and couple to isomerization reactions in molecular detail. In particular, we analyze the angular correlation functions of the backbone N-H bonds and find that slow conformational transitions occur at about the 310 helix in the apo state. These events at the helical region further affect the residues at about the ligand binding site. Unfolding of this helix leads to a tight hydrogen bond between the helical region and the ligand binding loop, thus forming a stable coiled structure. The helical and coiled structures are found to be characteristic of the Pin1-ligand complex with the ligand in the trans and cis states, respectively. These results indicate that the changes in the slow dynamics of Pin1 by the isomerization reaction occur via the shift in populations of the helical and coiled states, where the balance is dependent on the ligand isomerization states.

Structural and dynamic studies of DNA recognition by NF-κB p50 RHR homodimer using methyl NMR spectroscopy.

Protein dynamics involving higher-energy sparsely populated conformational substates are frequently critical for protein function. This study describes the dynamics of the homodimer (p50)2 of the p50 Rel homology region (RHR) of the transcription factor NF-κB, using 13C relaxation dispersion experiments with specifically (13C, 1H)-labeled methyl groups of Ile (δ), Leu and Val. Free (p50)2 is highly dynamic in solution, showing μs-ms relaxation dispersion consistent with exchange between the ground state and higher energy substates. These fluctuations propagate from the DNA-binding loops through the core of the domain. The motions are damped in the presence of κB DNA, but the NMR spectra of the DNA complexes reveal multiple local conformations of the p50 RHR homodimer bound to certain κB DNA sequences. Varying the length and sequence of κB DNA revealed two factors that promote a single bound conformation for the complex: the length of the κB site in the duplex and a symmetrical sequence of guanine nucleotides at both ends of the recognition motif. The dynamic nature of the DNA-binding loops, together with the multiple bound conformations of p50 RHR with certain κB sites, is consistent with variations in the transcriptional activity of the p50 homodimer with different κB sequences.

Inherent Minor Conformer of Bordetella Effector BteA Directs Chaperone-Mediated Unfolding.

The pathogen Bordetella pertussis uses a type-3 secretion system (T3SS) to inject its cytotoxic effector BteA into the host cell via a designated needle structure. Prior to injection BteA is bound to its cognate chaperone BtcA presumed to assist in effector unfolding en route to needle passage. We utilized NMR and EPR spectroscopy to uncover the molecular mechanism of BtcA-mediated unfolding of BteA. BtcA induces a global structural change in the effector, which adopts a more extended and partially unfolded conformation. EPR distance measurements further show that the structured helical-bundle form of free BteA exists in conformational equilibrium with a lowly populated minor species. The nature of this equilibrium was probed using NMR relaxation dispersion experiments. At 283 K structural effects are most pronounced for a contiguous surface spanning the A- and B-helices of BteA, extending at 303 K to a second surface including the D- and E-helices. Residues perturbed in the minor conformation coincide with those exhibiting a BtcA-induced increase in flexibility, identifying this conformation as the BtcA-bound form of the effector. Our findings hint at a conformational-selectivity mechanism for the chaperone interaction with the effector, a paradigm that may be common to effector-chaperones secretion complexes in this family of pathogens.

TAPBPR employs a ligand-independent docking mechanism to chaperone MR1 molecules.

Chaperones tapasin and transporter associated with antigen processing (TAP)-binding protein related (TAPBPR) associate with the major histocompatibility complex (MHC)-related protein 1 (MR1) to promote trafficking and cell surface expression. However, the binding mechanism and ligand dependency of MR1/chaperone interactions remain incompletely characterized. Here in vitro, biochemical and computational studies reveal that, unlike MHC-I, TAPBPR recognizes MR1 in a ligand-independent manner owing to the absence of major structural changes in the MR1 α2-1 helix between empty and ligand-loaded molecules. Structural characterization using paramagnetic nuclear magnetic resonance experiments combined with restrained molecular dynamics simulations reveals that TAPBPR engages conserved surfaces on MR1 to induce similar adaptations to those seen in MHC-I/TAPBPR co-crystal structures. Finally, nuclear magnetic resonance relaxation dispersion experiments using 19F-labeled diclofenac show that TAPBPR can affect the exchange kinetics of noncovalent metabolites with the MR1 groove, serving as a catalyst. Our results support a role of chaperones in stabilizing nascent MR1 molecules to enable loading of endogenous or exogenous cargo.