Effect of monovalent ion binding on molecular dynamics of the S100-family calcium-binding protein calbindin D9k.

Calbindin D9k is a member of the S100 subfamily of EF-hand calcium binding proteins, and has served as an important model system for biophysical studies. The fast timescale dynamics of the calcium-free (apo) state is characterized using molecular dynamics simulations. Order parameters for the backbone NH bond vectors are determined from the simulations and compared with experimentally derived values, with a focus on the dynamics of calcium-binding site I. There is a significant discrepancy between simulated and experimental order parameters for site I residues in the case of no ion bound in site I. However, it was found in the simulations that a Na+ ion can bind in site I, and the resulting order parameters determined from the simulations are in excellent agreement with experiment. Comparisons are made to X-ray structures of other S100 family members in which Na+ ions were observed or suggested to be bound in site I. © 2019 Wiley Periodicals, Inc.

Enhanced spectral density mapping through combined multiple-field deuterium 13CH2D methyl spin relaxation NMR spectroscopy.

Quadrupolar relaxation of 2H (D) nuclear spins is a powerful probe of conformational dynamics in biological macromolecules. Deuterium relaxation rate constants are determined by the spectral density function for reorientation of the C-D bond vector at zero, single-quantum, and double-quantum 2H frequencies. In the present work, 2H relaxation rate constants were measured for an E. coli ribonuclease H [U-2H, 15N] ILV-[13CH2D] sample using 400, 500, 800, and 900 MHz NMR spectrometers and analyzed by three approaches to determine spectral density values. First, data recorded at each static magnetic field were analyzed independently. Second, data recorded at 400 and 800 MHz were analyzed jointly and data recorded at other fields were analyzed independently. Third, data recorded at 400 and 500 MHz were interpolated to 450 MHz, and the resulting two pairs of data, corresponding to 400 MHz/800 MHz and 450 MHz/900 MHz, were analyzed jointly. The second and third approaches rely on the identity between the double quantum frequency at the lower field and the single quantum frequency at the higher field. Spectral density values for 32 of the 48 resolvable ILV methyl resonances were fit by the Lipari-Szabo model-free formalism and used to validate the three methods. The three spectral density mapping methods performed equally well in cross validation with data recorded at 700 MHz. However, the third method yielded approximately 10-15% more precise estimates of model-free parameters and consequently provides a general strategy for analysis of 2H spin relaxation data in biological macromolecules.

General Expressions for Carr-Purcell-Meiboom-Gill Relaxation Dispersion for N-Site Chemical Exchange.

The Carr-Purcell-Meiboom-Gill (CPMG) nuclear magnetic resonance experiment is widely used to characterize chemical exchange phenomena in biological macromolecules. Theoretical expressions for the nuclear spin relaxation rate constant for two-site chemical exchange during CPMG pulse trains valid for all time scales are well-known as are descriptions of N-site exchange in the fast limit. We have obtained theoretical expressions for N-site exchange outside of the fast limit by using approximations to an average Liouvillian describing the decay of magnetization during a CPMG pulse train. We obtain general expressions for CPMG experiments for any N-site scheme and all experimentally accessible time scales. For sufficiently slow chemical exchange, we obtain closed-form expressions for the relaxation rate constant and a general characteristic polynomial for arbitrary kinetic schemes. Furthermore, we highlight features that qualitatively characterize CPMG curves obtained for various N-site kinetic topologies, quantitatively characterize CPMG curves obtained from systems in various N-site exchange situations, and test distinguishability of kinetic models.

Lysine Side-Chain Dynamics in the Binding Site of Homeodomain/DNA Complexes As Observed by NMR Relaxation Experiments and Molecular Dynamics Simulations.

An important but poorly characterized contribution to the thermodynamics of protein-DNA interactions is the loss of entropy that occurs from restricting the conformational freedom of amino acid side chains. The effect of restricting the flexibility of several side chains at a protein-DNA interface may be comparable in many cases to the other factors that determine the binding thermodynamics and may, therefore, play a key role in dictating the binding affinity and/or specificity. Because the entropic contributions, including the presence and influence of side-chain dynamics, are especially difficult to estimate based on structural information, it is important to pursue experimental and theoretical studies that can provide direct information regarding these issues. We report on studies of a model system, the homeodomain/DNA complex, focusing on the Lys50 class of homeodomains where a key lysine residue in position 50 was shown previously to be critical for binding site specificity. NMR methodology was employed for determining the dynamics of lysine side-chain amino groups via 15N relaxation measurements in the Lys50-class homeodomains from the Drosophila protein Bicoid and the human protein Pitx2. In the case of Pitx2, complexes with both a consensus and a nonconsensus DNA binding site were examined. NMR-derived order parameters indicated moderate to substantial conformational freedom for the lysine NH3+ group in the complexes studied. To complement the experimental NMR measurements, molecular dynamics simulations were performed for the consensus complexes to gain further, detailed insights regarding the dynamics of the Lys50 side chain and other important residues in the protein-DNA interface.

Effects of interleukin-10 on cutaneous wounds and scars in humans of African continental ancestral origin.

Scars in humans of African continental ancestry heal with an exaggerated inflammatory response and a generally wider scar. Interleukin-10 is an anti-inflammatory and antifibrotic cytokine. A randomized controlled trial in Caucasians found that exogenous interleukin-10 resulted in improved macroscopic scar appearance and reduced scar redness. We investigated the effects of interleukin-10 on cutaneous scarring in volunteers of African ancestral origin in an exploratory, single-center, within-subject, double-blind randomized controlled trial. Fifty-six subjects received two of four potential prerandomized concentrations of interleukin-10 (5, 25, 100, and 250 ng/100 µL) in two full-thickness incisions on the upper inner arms. Anatomically matching incisions on the contralateral arm were treated with placebo. Scars were excised at 1 month for histological analysis and were redosed with the same regimen. Resultant excision scars were followed up for 12 months for scar width measurement and scoring. Scoring was performed by trial doctors, subjects, and a panel. Incisions treated with 100 ng/100 µL interleukin-10 had significantly reduced microscopic scar widths. Incisions treated with 5 and 25 ng/100 µL interleukin-10 were also narrower, but not significantly. There were no differences observed in pro-inflammatory or pro-fibrotic markers between interleukin-10 and placebo treatment. There was no long-term evidence that 100 ng/100 µL interleukin-10 had a therapeutic effect on macroscopic scar width or appearance, as excisions treated with this concentration were significantly wider than placebo between 8 and 12 months of maturation. Doctors showed a trend toward favoring the macroscopic appearance of placebo-treated excisions compared with those treated with 250 ng/100 µL interleukin-10. Panelists scored placebo-treated excisions as significantly better-appearing than those treated with 250 ng/100 µL interleukin-10. Doctors' scores showed a trend toward favoring treatment with 5 ng/100 µL interleukin-10 at 10 and 11 months post-excision. Subjects showed a trend toward favoring treatment with 5 ng/100 µL interleukin-10 between 5 and 9 months postexcision. Analysis of images of markedly improved scars revealed a potential subset of responders among those treated with 5 ng/100 µL interleukin-10. No concentration of interleukin-10 produced a statistically significant improvement in scarring compared with placebo.

The N-terminal basolateral targeting signal unlikely acts alone in the differential trafficking of membrane transporters in MDCK cells.

We have shown previously, using confocal imaging and transport assays, that the N-terminus of sodium-dependent vitamin C transporter 2 (SVCT2) can redirect apical SVCT1 to the basolateral membrane. Here, the SVCT model was used to further characterize the basolateral targeting peptide signal. Both the length (31 amino acids) and sequence accuracy of the N-terminus of SVCT2 were found to be important in basolateral targeting activity, suggesting a structural requirement. However, the N-terminal basolateral targeting sequence did not appear to act alone, based on analyses of heterologous chimeras. Although diverse N-terminal basolateral targeting signals from multipass membrane proteins can all redirect apical protein from the same gene family to the basolateral membrane, none of the N-terminal basolateral targeting signals can redirect the transmembrane and C-terminal regions from a different gene family. Instead, the presence of these heterologous N-terminal basolateral targeting signals affected the trafficking of otherwise apical protein, causing their accumulation in a stable tubulin-like non-actin structure. Nontargeting N-terminal sequences had no effect. Similar protein retention was observed previously and in this study when the C-terminus of apical or basolateral protein was mutated. These results suggest that the N- and C-termini interact, directly or indirectly, within each gene family for basolateral targeting. Circular dichroism and two-dimensional nuclear magnetic resonance analyses both found a lack of regular secondary structure in the conserved N-terminus of SVCT2, consistent with the presence of partner(s) in the targeting unit. Our finding, a departure from the prevailing single-peptide motif model, is consistent with the evolution of basolateral transporters from the corresponding apical genes. The interaction among the N-terminus, its partner(s), and the cellular basolateral targeting machinery needs to be further elucidated.

Structural and biophysical insights into the ligand-free Pitx2 homeodomain and a ring dermoid of the cornea inducing homeodomain mutant.

The homeodomain-containing transcription factor Pitx2 (pituitary homeobox protein 2) is present in many developing embryonic tissues, including the heart. Its homeodomain is responsible for the recognition and binding to target DNA sequences and thus constitutes a major functional unit in the Pitx2 protein. Nuclear magnetic resonance techniques were employed to determine the solution structure of the native Pitx2 homeodomain and a R24H mutant that causes autosomal dominantly inherited ring dermoid of the cornea syndrome. The structures reveal that both isoforms possess the canonical homeodomain fold. However, the R24H mutation results in a 2-fold increase in DNA binding affinity and a 5 °C decrease in thermal stability, while changing the dynamic environment of the homeodomain only locally. When introduced into full-length Pitx2c, the mutation results in an only 25% loss of transactivation activity. Our data correlate well with clinical observations suggesting a milder deficiency for the R24H mutation compared to those of other Pitx2 homeodomain mutations.

Observing selected domains in multi-domain proteins via sortase-mediated ligation and NMR spectroscopy.

NMR spectroscopy has distinct advantages for providing insight into protein structures, but faces significant resolution challenges as protein size increases. To alleviate such resonance overlap issues, the ability to produce segmentally labeled proteins is beneficial. Here we show that the S. aureus transpeptidase sortase A can be used to catalyze the ligation of two separately expressed domains of the same protein, MecA (B. subtilis). The yield of purified, segmentally labeled MecA protein conjugate is approximately 40%. The resultant HSQC spectrum obtained from this domain-labeled conjugate demonstrates successful application of sortase A for segmental labeling of multi-domain proteins for solution NMR study.

A qualitative study on the impact of caring for an ambulatory individual with nonsense mutation Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy is a rare genetic neuromuscular disorder, which can result in early death due to disease progression. Ataluren is indicated for the treatment of nonsense mutation Duchenne muscular dystrophy, in ambulatory individuals aged two years and older. This study explored the impact of caring for an ambulatory individual with nonsense mutation Duchenne muscular dystrophy, as well as the impact of treatment with ataluren on the caregiver experience, using retrospective recall. METHODS: Qualitative interviews were conducted with caregivers in the UK. Interviews were conducted by telephone, were recorded and transcribed. Data were analysed using thematic analysis and saturation was recorded. RESULTS: Ten interviews were conducted with parents of individuals aged 4-19 years. Caregivers reported proximal impacts (physical, emotional, time-related), and distal impacts (work, relationships, social life) of caring for their sons. The relationships between these impacts were illustrated in a conceptual model. Changes to the caregiver experience since initiation with their son's treatment were discussed. CONCLUSION: Caring for an ambulatory individual with nonsense mutation Duchenne muscular dystrophy has a substantial multifaceted impact on caregivers. Treatments which have the potential to improve symptoms or delay progression, may also have a positive impact on the quality of life of caregivers.

Symptoms and impacts of ambulatory nonsense mutation Duchenne muscular dystrophy: a qualitative study and the development of a patient-centred conceptual model.

BACKGROUND: Duchenne muscular dystrophy is a rare genetic neuromuscular disorder, which can result in early death due to disease progression. Ataluren is indicated for the treatment of nonsense mutation Duchenne muscular dystrophy, in ambulatory individuals aged two years and older. This study explored the symptoms and impacts of nonsense mutation Duchenne muscular dystrophy and experience with ataluren. METHODS: Qualitative interviews were conducted with caregivers in the UK. Interviews were conducted by telephone, were recorded and transcribed. Data were analysed using thematic analysis and saturation was recorded. RESULTS: Ten interviews were conducted with parents of individuals aged 4-19 years. Key symptoms included muscle weakness and muscle breakdown, which were associated with limitations in physical function and pain. These impacted individuals' daily activities, social activities and emotional wellbeing. These concepts and relationships were illustrated in a conceptual model, along with positive and negative moderating factors. Experience with ataluren and changes since initiation with treatment were discussed. CONCLUSION: Individuals with nonsense mutation Duchenne muscular dystrophy experience a range of interrelated symptoms and functional issues which impact their broader health-related quality of life. Treatments which address this high unmet need have the potential to improve the health-related quality of life of these individuals.

Insights into the nature of DNA binding of AbrB-like transcription factors.

Understanding the DNA recognition and binding by the AbrB-like family of transcriptional regulators is of significant interest since these proteins enable bacteria to elicit the appropriate response to diverse environmental stimuli. Although these "transition-state regulator" proteins have been well characterized at the genetic level, the general and specific mechanisms of DNA binding remain elusive. We present RDC-refined NMR solution structures and dynamic properties of the DNA-binding domains of three Bacillus subtilis transition-state regulators: AbrB, Abh, and SpoVT. We combined previously investigated DNase I footprinting, DNA methylation, gel-shift assays, and mutagenic and NMR studies to generate a structural model of the complex between AbrBN(55) and its cognate promoter, abrB8. These investigations have enabled us to generate a model for the specific nature of the transition-state regulator-DNA interaction, a structure that has remained elusive thus far.

Compact expressions for R1ρ relaxation for N-site chemical exchange using Schur decomposition.

The rotating-frame spin relaxation rate constant, R1ρ, is a powerful probe of macromolecular chemical and conformational dynamics in relaxation dispersion, CEST, and DEST NMR experiments. The R1ρ relaxation rate constant is given by the absolute value of the largest (least negative) eigenvalue of the Bloch-McConnell evolution matrix; however, estimation of this eigenvalue require inversion of 3 N × 3 N dimensional matrices, in which N is the number of interconverting sites or states for a given nuclear spin in a molecule. The Schur complement is used to reduce the problem of calculating the characteristic polynomial of a 3 N × 3 N matrix to that of calculating the characteristic polynomial of a 3 × 3 matrix. The resulting expressions for N-site chemical exchange are more numerically tractable, because the largest matrix inversion also is of dimension 3 × 3. In addition, the simplifications offered by the Schurr complement conveniently illustrate the effects of fast or slow kinetic steps within an N-site kinetic topology.

Algebraic expressions for Carr-Purcell-Meiboom-Gill relaxation dispersion for N-site chemical exchange.

The Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiment measures the effective relaxation rate constant during a train of spin-echo pulse sequence elements as a function of the echo time. The CPMG experiment is a powerful method for characterizing chemical and conformational dynamic processes, termed chemical and conformational exchange, on µs-ms time scales, comparable to the experimentally accessible echo times. Approximate theoretical expressions for the effective relaxation rate constant for N-site chemical exchange have been reported (H. Koss, M. Rance, and A. G. Palmer, Biochemistry 57, 4753-4763 (2018)). Expressions for the effective relaxation rate constant have been improved by using the Cayley-Hamilton theorem to obtain simple and accurate approximations of the average Liouvillian for the CPMG experiment. The improved accuracy of the results allows efficient analyses of experimental data. In addition, the relationship is clarified between the approach of Koss and coworkers and that of Jen (1978).

Protein side-chain dynamics and residual conformational entropy.

Changes in residual conformational entropy of proteins can be significant components of the thermodynamics of folding and binding. Nuclear magnetic resonance (NMR) spin relaxation is the only experimental technique capable of probing local protein entropy, by inference from local internal conformational dynamics. To assess the validity of this approach, the picosecond-to-nanosecond dynamics of the arginine side-chain N(epsilon)-H(epsilon) bond vectors of Escherichia coli ribonuclease H (RNase H) were determined by NMR spin relaxation and compared to the mechanistic detail provided by molecular dynamics (MD) simulations. The results indicate that arginine N(epsilon) spin relaxation primarily reflects persistence of guanidinium salt bridges and correlates well with simulated side-chain conformational entropy. In particular cases, the simulations show that the aliphatic part of the arginine side chain can retain substantial disorder while the guanidinium group maintains its salt bridges; thus, the N(epsilon)-H(epsilon) bond-vector orientation is conserved and side-chain flexibility is concealed from N(epsilon) spin relaxation. The MD simulations and an analysis of a rotamer library suggest that dynamic decoupling of the terminal moiety from the remainder of the side chain occurs for all five amino acids with more than two side-chain dihedral angles (R, K, E, Q, and M). Dynamic decoupling thus may represent a general biophysical strategy for minimizing the entropic penalties of folding and binding.

Defining a Canonical Ligand-Binding Pocket in the Orphan Nuclear Receptor Nurr1.

Nuclear receptor-related 1 protein (Nurr1/NR4A2) is an orphan nuclear receptor (NR) that is considered to function without a canonical ligand-binding pocket (LBP). A crystal structure of the Nurr1 ligand-binding domain (LBD) revealed no physical space in the conserved region where other NRs with solvent accessible apo-protein LBPs bind synthetic and natural ligands. Using solution nuclear magnetic resonance spectroscopy, hydrogen/deuterium exchange mass spectrometry, and molecular dynamics simulations, we show that the putative canonical Nurr1 LBP is dynamic with high solvent accessibility, exchanges between two or more conformations on the microsecond-to-millisecond timescale, and can expand from the collapsed crystallized conformation to allow binding of unsaturated fatty acids. These findings should stimulate future studies to probe the ligandability and druggability of Nurr1 for both endogenous and synthetic ligands, which could lead to new therapeutics for Nurr1-related diseases, including Parkinson's disease and schizophrenia.

Temperature dependence of the NMR generalized order parameter.

The study of protein conformational dynamics is motivated in large part by a desire to understand the forces present at different sites throughout the molecular structure. The generalized order parameter determined by NMR spectroscopy has played a central role in the study of protein dynamics on the picosecond-nanosecond time scale. A modeling procedure is presented for analysis of the temperature dependence of the generalized order parameter that extends a previous analysis (Massi and Palmer, J Am Chem Soc 2003;125:11158-11159). As part of this procedure, the potential of mean force is characterized for the N-H bond vectors of the protein backbone. This procedure accounts for the observed temperature dependence of the generalized order parameter in a representative data set from the B1 domain of Streptococcal protein G (Seewald, Pichumani, Stowell, Tibbals, Regan, and Stone, Protein Sci 2000;9:1177-1193). The results indicate a general trend, in which the force constants associated with the potential of mean force decrease with increasing temperature. The analysis also provides evidence for variations in the potential of mean force for different secondary structural elements.

Effects of calcium binding on the side-chain methyl dynamics of calbindin D9k: a 2H NMR relaxation study.

The effects of Ca(2+) binding on the side-chain methyl dynamics of calbindin D(9k) have been characterized by (2)H NMR relaxation rate measurements. Longitudinal, transverse in-phase, quadrupolar order, transverse anti-phase and double quantum relaxation rates are reported for both the apo and Ca(2+)-loaded states of the protein at two magnetic field strengths. The relatively large size of the data set allows for a detailed analysis of the underlying conformational dynamics by spectral density mapping and model-free fitting procedures. The results reveal a correlation between a methyl group's distance from the Ca(2+) binding sites and its conformational dynamics. Several methyl groups segregate into two limiting classes, one proximal and the other distal to the binding sites. Methyl groups in these two classes respond differently to Ca(2+) binding, both in terms of the timescale and amplitude of their fluctuations. Ca(2+) binding elicits a partial immobilization among methyl groups in the proximal class, which is consistent with previous studies of calbindin's backbone dynamics. The distal class, however, exhibits a trend that could not be inferred from the backbone data in that its mobility actually increases with Ca(2+) binding. We have introduced the term polar dynamics to describe this type of organization across the molecule. The trend may represent an important mechanism by which calbindin D(9k) achieves high affinity binding while minimizing the corresponding loss of conformational entropy.

The solution structure of the native K50 Bicoid homeodomain bound to the consensus TAATCC DNA-binding site.

The solution structure of the homeodomain of the Drosophila morphogenic protein Bicoid (Bcd) complexed with a TAATCC DNA site is described. Bicoid is the only known protein that uses a homeodomain to regulate translation, as well as transcription, by binding to both RNA and DNA during early Drosophila development; in addition, the Bcd homeodomain can recognize an array of different DNA sites. The dual functionality and broad recognition capabilities signify that the Bcd homeodomain may possess unique structural/dynamic properties. Bicoid is the founding member of the K50 class of homeodomain proteins, containing a lysine residue at the critical 50th position (K50) of the homeodomain sequence, a residue required for DNA and RNA recognition; Bcd also has an arginine residue at the 54th position (R54), which is essential for RNA recognition. Bcd is the only known homeodomain with the K50/R54 combination of residues. The Bcd structure indicates that this homeodomain conforms to the conserved topology of the homeodomain motif, but exhibits a significant variation from other homeodomain structures at the end of helix 1. A key result is the observation that the side-chains of the DNA-contacting residues K50, N51 and R54 all show strong signs of flexibility in the protein-DNA interface. This finding is supportive of the adaptive-recognition theory of protein-DNA interactions.