| 1. |
- Han, X., et al.
(författare)
-
Assignment of IVL-Methyl side chain of the ligand-free monomeric human MALT1 paracaspase-IgL(3) domain in solution
- 2022
-
Ingår i: Biomolecular Nmr Assignments. - : Springer Science and Business Media LLC. - 1874-2718 .- 1874-270X. ; 16:2, s. 363-371
-
Tidskriftsartikel (refereegranskat)abstract
- Mucosa-associated lymphoid tissue protein 1 (MALT1) plays a key role in adaptive immune responses by modulating specific intracellular signalling pathways that control the development and proliferation of both T and B cells. Dysfunction of these pathways is coupled to the progress of highly aggressive lymphoma as well as to potential development of an array of different immune disorders. In contrast to other signalling mediators, MALT1 is not only activated through the formation of the CBM complex together with the proteins CARMA1 and Bcl10, but also by acting as a protease that cleaves multiple substrates to promote lymphocyte proliferation and survival via the NF-kappa B signalling pathway. Herein, we present the partial H-1, C-13 Ile/Val/Leu-Methyl resonance assignment of the monomeric apo form of the paracaspase-IgL(3) domain of human MALT1. Our results provide a solid ground for future elucidation of both the three-dimensional structure and the dynamics of MALT1, key for adequate development of inhibitors, and a thorough molecular understanding of its function(s).
|
|
| 2. |
- Manzoni, Francesco, et al.
(författare)
-
Elucidation of Hydrogen Bonding Patterns in Ligand-Free, Lactose- and Glycerol-Bound Galectin-3C by Neutron Crystallography to Guide Drug Design
- 2018
-
Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 1520-4804 .- 0022-2623. ; 61:10, s. 4412-4420
-
Tidskriftsartikel (refereegranskat)abstract
- The medically important drug target galectin-3 binds galactose-containing moieties on glycoproteins through an intricate pattern of hydrogen bonds to a largely polar surface-exposed binding site. All successful inhibitors of galectin-3 to date have been based on mono- or disaccharide cores closely resembling natural ligands. A detailed understanding of the H-bonding networks in these natural ligands will provide an improved foundation for the design of novel inhibitors. Neutron crystallography is an ideal technique to reveal the geometry of hydrogen bonds because the positions of hydrogen atoms are directly detected rather than being inferred from the positions of heavier atoms as in X-ray crystallography. We present three neutron crystal structures of the C-terminal carbohydrate recognition domain of galectin-3: the ligand-free form and the complexes with the natural substrate lactose and with glycerol, which mimics important interactions made by lactose. The neutron crystal structures reveal unambiguously the exquisite fine-tuning of the hydrogen bonding pattern in the binding site to the natural disaccharide ligand. The ligand-free structure shows that most of these hydrogen bonds are preserved even when the polar groups of the ligand are replaced by water molecules. The protonation states of all histidine residues in the protein are also revealed and correlate well with NMR observations. The structures give a solid starting point for molecular dynamics simulations and computational estimates of ligand binding affinity that will inform future drug design.
|
|
| 3. |
- Verteramo, Maria Luisa, et al.
(författare)
-
Interplay of halogen bonding and solvation in protein-ligand binding
- 2024
-
Ingår i: iScience. - 2589-0042. ; 27:4
-
Tidskriftsartikel (refereegranskat)abstract
- Halogen bonding is increasingly utilized in efforts to achieve high affinity and selectivity of molecules designed to bind proteins, making it paramount to understand the relationship between structure, dynamics, and thermodynamic driving forces. We present a detailed analysis addressing this problem using a series of protein-ligand complexes involving single halogen substitutions - F, Cl, Br, and I - and nearly identical structures. Isothermal titration calorimetry reveals an increasingly favorable binding enthalpy from F to I that correlates with the halogen size and σ-hole electropositive character, but is partially counteracted by unfavorable entropy, which is constant from F to Cl and Br, but worse for I. Consequently, the binding free energy is roughly equal for Cl, Br, and I. QM and solvation-free-energy calculations reflect an intricate balance between halogen bonding, hydrogen bonds, and solvation. These advances have the potential to aid future drug design initiatives involving halogenated compounds.
|
|
| 4. |
- Wallerstein, Johan, et al.
(författare)
-
Entropy-Entropy Compensation between the Protein, Ligand, and Solvent Degrees of Freedom Fine-Tunes Affinity in Ligand Binding to Galectin-3C
- 2021
-
Ingår i: Jacs Au. - : American Chemical Society (ACS). - 2691-3704. ; 1:4, s. 484-500
-
Tidskriftsartikel (refereegranskat)abstract
- Molecular recognition is fundamental to biological signaling. A central question is how individual interactions between molecular moieties affect the thermodynamics of ligand binding to proteins and how these effects might propagate beyond the immediate neighborhood of the binding site. Here, we investigate this question by introducing minor changes in ligand structure and characterizing the effects of these on ligand affinity to the carbohydrate recognition domain of galectin-3, using a combination of isothermal titration calorimetry, X-ray crystallography, NMR relaxation, and computational approaches including molecular dynamics (MD) simulations and grid inhomogeneous solvation theory (GIST). We studied a congeneric series of ligands with a fluorophenyl-triazole moiety, where the fluorine substituent varies between the ortho, meta, and para positions (denoted O, M, and P). The M and P ligands have similar affinities, whereas the O ligand has 3-fold lower affinity, reflecting differences in binding enthalpy and entropy. The results reveal surprising differences in conformational and solvation entropy among the three complexes. NMR backbone order parameters show that the O-bound protein has reduced conformational entropy compared to the M and P complexes. By contrast, the bound ligand is more flexible in the O complex, as determined by F-19 NMR relaxation, ensemble-refined X-ray diffraction data, and MD simulations. Furthermore, GIST calculations indicate that the O-bound complex has less unfavorable solvation entropy compared to the other two complexes. Thus, the results indicate compensatory effects from ligand conformational entropy and water entropy, on the one hand, and protein conformational entropy, on the other hand. Taken together, these different contributions amount to entropy-entropy compensation among the system components involved in ligand binding to a target protein.
|
|
| 5. |
- Wallerstein, Johan, et al.
(författare)
-
Minute Additions of DMSO Affect Protein Dynamics Measurements by NMR Relaxation Experiments through Significant Changes in Solvent Viscosity
- 2019
-
Ingår i: ChemPhysChem. - : Wiley. - 1439-4235. ; 20:2, s. 326-332
-
Tidskriftsartikel (refereegranskat)abstract
- Studies of protein−ligand binding often rely on dissolving the ligand in dimethyl sulfoxide (DMSO) to achieve sufficient solubility, and then titrating the ligand solution into the protein solution. As a result, the final protein−ligand solution contains small amounts of DMSO in the buffer. Here we report how the addition of DMSO impacts studies of protein conformational dynamics. We used 15N NMR relaxation to compare the rotational diffusion correlation time (τC) of proteins in aqueous buffer with and without DMSO. We found that τC scales with the viscosity of the water−DMSO mixture, which depends sensitively on the amount of DMSO and varies by a factor of 2 across the relevant concentration range. NMR relaxation studies of side chains dynamics are commonly interpreted using τC as a fixed parameter, obtained from backbone 15N relaxation data acquired on a separate sample. Model-free calculations show that errors in τC, arising from mismatched DMSO concentration between samples, lead to significant errors in order parameters. Our results highlight the importance of determining τC for each sample or carefully matching the DMSO concentrations between samples.
|
|
| 6. |
- Wallerstein, Johan
(författare)
-
Molecular recognition and dynamics in proteins studied by NMR
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Knowledge of dynamics in protein is very important in the description of protein function and molecular recognition. The thesis investigates protein dynamics on time-scales from milli- to sub-nanosecond, with focus on the latter, using NMR spin relaxation experiments on two proteins, the 138-residue carbohydrate recognition domain of galectin-3 (Gal3C) and the 56-residue B1 domain of bacterial protein G (PGB1). Fives studies are presented.By measuring the exchange contribution to R2-relaxation, as a function of pH, using 13C-CPMG-experiments directed on the side-chain carbonyls of glutamic and aspartic acid in PGB1, we could determine site specific protonation rate constants k(on), and deprotonation rate constants k(off). A linear free-energy relationship between log k(on) and pKa is found, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these rates arises from charge stabilization of the deprotonated state.Dimethyl sulfoxide (DMSO) is often used for dissolving nonpolar ligands in protein-ligand studies. DMSO change the viscosity, which is proportional to rotational correlation time. The correlation time of PGB1 and Gal3C was determined for different DMSO-concentrations. Effects of minute additions of DMSO in samples used in spin-relaxation experiments were examined for the case of 2H-methyl side chain model-free studies. Chemical shift perturbations studies on apo-Gal3C show uniform changes of chemical shifts, indicating DMSO has a minor impact on the hydration layer.15N-backbone and 2H-methyl side chain NMR order parameters (S2) are determined for three Gal3C complexes using the Lipari-Szabo model-free formalism developed in the 80s. Minor changes in ligand structure generate differences in the conformational entropy. Specifically, the radial distribution of conformational entropy, with ligand in the centre reveals how entropy varies between consecutive shells within the protein. The study combines NMR relaxation with isothermal titration calorimetry (ITC), X-ray crystallography, and computational approaches.The structure–thermodynamic relationship for halogen bonds C–X (X=F, Cl, Br, and I) between the ligand substituents and the backbone C=O of a glycine in Gal3C was studied with NMR, ITC, X-ray crystallography and computational approaches. The sigma-hole associated with the halogen bond affects the electron density of surrounding 15N nuclei in amides, and consequently the chemical shift of these nuclei. There is a correlation between ITC-determined enthalpy of binding vs amide chemical shift.In Gal3C bound to natural substrate lactose, the chemical shifts changes of the 13C-E1 in histidine side chains are traced as a function of pH using 1H-13C HSQC-experiments. The experiment helps in determination of the tautomeric state in the proteins four histidines. The histidine in the binding site is in tautomer ND1, another is in tautomer NE2 and two are partly charged. Neutron diffraction studies at pH 7.5 confirms the NMR-studies.
|
|
| 7. |
- Wallerstein, Johan, et al.
(författare)
-
Site-Specific Protonation Kinetics of Acidic Side Chains in Proteins Determined by pH-Dependent Carboxyl (13)C NMR Relaxation.
- 2015
-
Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 1520-5126 .- 0002-7863. ; 137:8, s. 3093-3101
-
Tidskriftsartikel (refereegranskat)abstract
- Proton-transfer dynamics plays a critical role in many biochemical processes, such as proton pumping across membranes and enzyme catalysis. The large majority of enzymes utilize acid-base catalysis and proton-transfer mechanisms, where the rates of proton transfer can be rate limiting for the overall reaction. However, measurement of proton-exchange kinetics for individual side-chain carboxyl groups in proteins has been achieved in only a handful of cases, which typically have involved comparative analysis of mutant proteins in the context of reaction network modeling. Here we describe an approach to determine site-specific protonation and deprotonation rate constants (kon and koff, respectively) of carboxyl side chains, based on (13)C NMR relaxation measurements as a function of pH. We validated the method using an extensively studied model system, the B1 domain of protein G, for which we measured rate constants koff in the range (0.1-3) × 10(6) s(-1) and kon in the range (0.6-300) × 10(9) M(-1) s(-1), which correspond to acid-base equilibrium dissociation constants (Ka) in excellent agreement with previous results determined by chemical shift titrations. Our results further reveal a linear free-energy relationship between log kon and pKa, which provides information on the free-energy landscape of the protonation reaction, showing that the variability among residues in these parameters arises primarily from the extent of charge stabilization of the deprotonated state by the protein environment. We find that side-chain carboxyls with extreme values of koff or kon are involved in hydrogen bonding, thus providing a mechanistic explanation for the observed stabilization of the protonated or deprotonated state.
|
|
