[Show abstract][Hide abstract] ABSTRACT: Merozoite surface protein 1 (MSP1) has been identified as a target antigen for protective immune responses against asexual blood stage malaria, but effective vaccines based on MSP1 have not been developed so far. We have modified the sequence of Plasmodium yoelii MSP119 (the C-terminal region of the molecule) and examined the ability of the variant proteins to bind protective monoclonal antibodies and to induce protection by immunization. In parallel, we examined the structure of the protein and the consequences of the amino acid changes. Naturally occurring sequence polymorphisms reduced the binding of individual protective antibodies, indicating that they contribute to immune evasion, but immunization with these variant proteins still provided protective immunity. One variant that resulted in the localized distortion of a loop close to the N-terminus of MSP119 almost completely ablated protection by immunization, indicating the importance of this region of MSP119 as a target for protective immunity and in vaccine development.
[Show abstract][Hide abstract] ABSTRACT: The discovery of effective new antimalarial agents is urgently needed. One of the most frequently studied molecules anchored
to the parasite surface is the merozoite surface protein-1 (MSP1). At red blood cell invasion MSP1 is proteolytically processed,
and the 19-kDa C-terminal fragment (MSP119) remains on the surface and is taken into the red blood cell, where it is transferred to the food vacuole and persists until
the end of the intracellular cycle. Because a number of specific antibodies inhibit erythrocyte invasion and parasite growth,
MSP119 is therefore a promising target against malaria. Given the structural homology of cupredoxins with the Fab domain of monoclonal
antibodies, an approach combining NMR and isothermal titration calorimetry (ITC) measurements with docking calculations based
on BiGGER is employed on MSP119-cupredoxin complexes. Among the cupredoxins tested, rusticyanin forms a well defined complex with MSP119 at a site that overlaps with the surface recognized by the inhibitory antibodies. The addition of holo-rusticyanin to infected
cells results in parasitemia inhibition, but negligible effects on parasite growth can be observed for apo-rusticyanin and
other proteins of the cupredoxin family. These findings point to rusticyanin as an excellent therapeutic tool for malaria
treatment and provide valuable information for drug design.
Preview · Article · Jun 2013 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: The high-resolution NMR structure of the N-domain of human eRF1, responsible for stop codon recognition, has been determined in solution. The overall fold of the protein is the same as that found in the crystal structure. However, the structures of several loops, including those participating in stop codon decoding, are different. Analysis of the NMR relaxation data reveals that most of the regions with the highest structural discrepancy between the solution and solid states undergo internal motions on the ps-ns and ms time scales. The NMR data show that the N-domain of human eRF1 exists in two conformational states. The distribution of the residues having the largest chemical shift differences between the two forms indicates that helices α2 and α3, with the NIKS loop between them, can switch their orientation relative to the β-core of the protein. Such structural plasticity may be essential for stop codon recognition by human eRF1.
[Show abstract][Hide abstract] ABSTRACT: In order to examine the origins of the large positive cooperativity (ΔG(0)(coop) = -2.9 kcal mol(-1)) of trimethoprim (TMP) binding to a bacterial dihydrofolate reductase (DHFR) in the presence of NADPH, we have determined and compared NMR solution structures of L. casei apo DHFR and its binary and ternary complexes with TMP and NADPH and made complementary thermodynamic measurements. The DHFR structures are generally very similar except for the A-B loop region and part of helix B (residues 15-31) which could not be directly detected for L. casei apo DHFR because of line broadening from exchange between folded and unfolded forms. Thermodynamic and NMR measurements suggested that a significant contribution to the cooperativity comes from refolding of apo DHFR on binding the first ligand (up to -0.95 kcals mol(-1) if 80% of A-B loop requires refolding). Comparisons of Cα-Cα distance differences and domain rotation angles between apo DHFR and its complexes indicated that generally similar conformational changes involving domain movements accompany formation of the binary complexes with either TMP or NADPH and that the binary structures are approaching that of the ternary complex as would be expected for positive cooperativity. These favorable ligand-induced structural changes upon binding the first ligand will also contribute significantly to the cooperative binding. A further substantial contribution to cooperative binding results from the proximity of the bound ligands in the ternary complex: this reduces the solvent accessible area of the ligand and provides a favorable entropic hydrophobic contribution (up to -1.4 kcal mol(-1)).
[Show abstract][Hide abstract] ABSTRACT: Termination of translation in eukaryotes is triggered by two polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3. eRF1 is a three-domain protein that interacts with eukaryotic class 2 polypeptide chain release factor 3 via its C-terminal domain (C-domain). The high-resolution NMR structure of the human C-domain (residues 277-437) has been determined in solution. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure. The structure of the minidomain (residues 329-372), which was ill-defined in the crystal structure, has been determined in solution. The protein backbone dynamics, studied using (15)N-relaxation experiments, showed that the C-terminal tail 414-437 and the minidomain are the most flexible parts of the human C-domain. The minidomain exists in solution in two conformational states, slowly interconverting on the NMR timescale. Superposition of this NMR solution structure of the human C-domain onto the available crystal structure of full-length human eRF1 shows that the minidomain is close to the stop codon-recognizing N-terminal domain. Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor. The results provide new insights into the possible role of the C-domain in the process of translation termination.
[Show abstract][Hide abstract] ABSTRACT: Translation termination in eukaryotes is governed by the interaction of two, class 1 and class 2, polypeptide chain release factors with the ribosome. The middle (M) domain of the class 1 factor eRF1 contains the strictly conserved GGQ motif and is involved in hydrolysis of the peptidyl-tRNA ester bond in the peptidyl transferase center of the large ribosome subunit. Heteronuclear NMR spectroscopy was used to map the interaction interface of the M domain of human eRF1 with eukaryotic ribosomes. The protein was found to specifically interact with the 60S subunit, since no interaction was detected with the 40S subunit. The amino acid residues forming the interface mostly belong to long helix alpha1 of the M domain. Some residues adjacent to alpha1 and belonging to strand beta5 and short helices alpha2 and alpha3 are also involved in the protein–ribosome contact. The functionally inactive G183A mutant interacted with the ribosome far more weakly as compared with the wild-type eRF1. The interaction interfaces of the two proteins were nonidentical. It was concluded that long helix alpha1 is functionally important and that the conformational flexibility of the GGQ loop is essential for the tight protein–ribosome contact.
Full-text · Article · Jun 2008 · Molecular Biology
[Show abstract][Hide abstract] ABSTRACT: We report NMR assignments of the protein backbone of the C-terminal domain (163 a.a.) of human class 1 translation termination factor eRF1. It was found that several protein loop residues exist in two slowly interconverting conformational states.
Full-text · Article · Jan 2008 · Biomolecular NMR Assignments
[Show abstract][Hide abstract] ABSTRACT: The eukaryotic class 1 polypeptide chain release factor is a three-domain protein involved in the termination of translation, the final stage of polypeptide biosynthesis. In attempts to understand the roles of the middle domain of the eukaryotic class 1 polypeptide chain release factor in the transduction of the termination signal from the small to the large ribosomal subunit and in peptidyl-tRNA hydrolysis, its high-resolution NMR structure has been obtained. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal. However, the orientation of the functionally critical GGQ loop and neighboring alpha-helices has genuine and noticeable differences in solution and in the crystal. Backbone amide protons of most of the residues in the GGQ loop undergo fast exchange with water. However, in the AGQ mutant, where functional activity is abolished, a significant reduction in the exchange rate of the amide protons has been observed without a noticeable change in the loop conformation, providing evidence for the GGQ loop interaction with water molecule(s) that may serve as a substrate for the hydrolytic cleavage of the peptidyl-tRNA in the ribosome. The protein backbone dynamics, studied using 15N relaxation experiments, showed that the GGQ loop is the most flexible part of the middle domain. The conformational flexibility of the GGQ and 215-223 loops, which are situated at opposite ends of the longest alpha-helix, could be a determinant of the functional activity of the eukaryotic class 1 polypeptide chain release factor, with that helix acting as the trigger to transmit the signals from one loop to the other.
[Show abstract][Hide abstract] ABSTRACT: The structure of the complex of the antibacterial drug trimethoprim with its target, the enzyme dihydrofolate reductase (DHFR)
of Lactobacillus casei, was studied using multidimensional NMR spectroscopy. Aseries of 33 high-resolution structures of this complex in solution
was calculated using the measured experimental parameters of the nuclear Overhauser effect, spin-spin coupling constants,
and residual dipolar coupling constants. The key interactions leading to stable binding of the drug with the enzyme were determined.
The resulting structural data were compared with structures of the ternary complex of trimethoprim and the coenzyme NADPH
with bacterial (L. casei) and human DHFR that were calculated previously. The most probable reasons for cooperative interaction of trimethoprim and
NADPH that determine the highly selective binding of the drug to the bacterial enzyme, in particular, the protein conformation
change on going from the binary to the ternary complex of DHFR, were discussed.
Full-text · Article · Jan 2007 · Pharmaceutical Chemistry Journal
[Show abstract][Hide abstract] ABSTRACT: SH3 domains are small protein modules that are involved in protein-protein interactions in several essential metabolic pathways. The availability of the complete genome and the limited number of clearly identifiable SH3 domains make the yeast Saccharomyces cerevisae an ideal proteomic-based model system to investigate the structural rules dictating the SH3-mediated protein interactions and to develop new tools to assist these studies. In the present work, we have determined the solution structure of the SH3 domain from Myo3 and modeled by homology that of the highly homologous Myo5, two myosins implicated in actin polymerization. We have then implemented an integrated approach that makes use of experimental and computational methods to characterize their binding properties. While accommodating their targets in the classical groove, the two domains have selectivity in both orientation and sequence specificity of the target peptides. From our study, we propose a consensus sequence that may provide a useful guideline to identify new natural partners and suggest a strategy of more general applicability that may be of use in other structural proteomic studies.
[Show abstract][Hide abstract] ABSTRACT: Amide protection factors have been determined from NMR measurements of hydrogen/deuterium amide NH exchange rates measured on assigned signals from Lactobacillus casei apo-DHFR and its binary and ternary complexes with trimethoprim (TMP), folinic acid and coenzymes (NADPH/NADP+). The substantial sizes of the residue-specific ΔH and TΔS values for the opening/closing events in NH exchange for most of the measurable residues in apo-DHFR indicate that sub-global or global rather than local exchange mechanisms are usually involved. The amide groups of residues in helices and sheets are those most protected in apo-DHFR and its complexes, and the protection factors are generally related to the tightness of ligand binding. The effects of ligand binding that lead to changes in amide protection are not localised to specific binding sites but are spread throughout the structure via a network of intramolecular interactions. Although the increase in protein stability in the DHFR.TMP.NADPH complex involves increased ordering in the protein structure (requiring TΔS energy) this is recovered, to a large extent, by the stronger binding (enthalpic ΔH) interactions made possible by the reduced motion in the protein. The ligand-induced protection effects in the ternary complexes DHFR.TMP.NADPH (large positive binding co-operativity) and DHFR.folinic acid.NADPH (large negative binding co-operativity) mirror the co-operative effects seen in the ligand binding. For the DHFR.TMP.NADPH complex, the ligand-induced protection factors result in ΔΔGo values for many residues being larger than the ΔΔGo values in the corresponding binary complexes. In contrast, for DHFR.folinic acid.NADPH, the ΔΔGo values are generally smaller than many of those in the corresponding binary complexes. The results indicate that changes in protein conformational flexibility on formation of the ligand complex play an important role in determining the co-operativity in the ligand binding.
[Show abstract][Hide abstract] ABSTRACT: All the aromatic proton resonances in the 500-MHzNMR spectra of Lactobacillus casei dihydrofolate reductase have been assigned for several of its complexes with inhibitors. For the complexes with methotrexate and trimethoprim this was achieved by using a combination of NMR techniques in conjunction with a selectively deuterated protein designed to simplify the spectra such that nuclear Overhauser effect (NOE) connections could be detected with greater ease and certainty. By correlating these NOE data with crystal structure data on related complexes it was possible to assign all the aromatic resonances and to extend these assignments to spectra of other complexes of dihydrofolate reductase.
The conformation-dependent chemical shifts observed for many of the resonances could be explained qualita-tively, but not quantitatively, in terms of ring-current shifts. The discrepancies between calculated ring-current shifts and the observed conformation-dependent shifts could not in general be accounted for satisfactorily in terms of carbonyl-group anisotropic shielding contributions calculated using presently available models. In the case of the Hδ1,δ2 protons of Phe30 some of the discrepancy probably results from a difference in the conformation of the Phe ring between the solution and crystal states.
[Show abstract][Hide abstract] ABSTRACT: Malarial merozoites invade erythrocytes; and as an essential step in this invasion process, the 42-kDa fragment of Plasmodium falciparum merozoite surface protein-1 (MSP142) is further cleaved to a 33-kDa N-terminal polypeptide (MSP133) and an 19-kDa C-terminal fragment (MSP119) in a secondary processing step. Suramin was shown to inhibit both merozoite invasion and MSP142 proteolytic cleavage. This polysulfonated naphthylurea bound directly to recombinant P. falciparum MSP142 (Kd = 0.2 μm) and to Plasmodium vivax MSP142 (Kd = 0.3 μm) as measured by fluorescence enhancement in the presence of the protein and by isothermal titration calorimetry. Suramin
bound only slightly less tightly to the P. vivax MSP133 (Kd = 1.5 μm) secondary processing product (fluorescence measurements), but very weakly to MSP119 (Kd ∼ 15 mm) (NMR measurements). Several residues in MSP119 were implicated in the interaction with suramin using NMR measurements. A series of symmetrical suramin analogues that differ
in the number of aromatic rings and substitution patterns of the terminal naphthylamine groups was examined in invasion and
processing assays. Two classes of analogue with either two or four bridging rings were found to be active in both assays,
whereas two other classes without bridging rings were inactive. We propose that suramin and related compounds inhibit erythrocyte
invasion by binding to MSP1 and by preventing its cleavage by the secondary processing protease. The results indicate that
enzymatic events during invasion are suitable targets for drug development and validate the novel concept of an inhibitor
binding to a macromolecular substrate to prevent its proteolysis by a protease.
Full-text · Article · Dec 2003 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: NKR-P1A is a C-type lectin-like receptor on natural killer cells believed to be involved in the cytotoxicity of these cells. Ligands for this protein are not known. Here, we describe the binding of a fully sulphated disaccharide, sucrose octasulphate, by the recombinant C-type lectin-like domain of NKR-P1A. The binding was observed by NMR spectroscopy methods that have recently been described for the screening of compound libraries for bioaffinities, namely the 2D NOESY and saturation transfer difference NMR experiments. (1)H titration studies indicate that the binding is specific. These findings raise the possibility that NKR-P1A recognises sulphated natural ligands in common with certain other members of the C-type lectin family.
[Show abstract][Hide abstract] ABSTRACT: Lactobacillus casei dihydrofolate reductase (DHFR) binds more than a thousand times tighter to NADPH than to NADP+. The origins of the difference in binding affinity to DHFR between NADPH and NADP+ are investigated in the present study using experimental NMR data and hybrid density functional, B3LYP, calculations. Certain protein residues (Ala 6, Gln 7, Ile 13 and Gly 14) that are directly involved in hydrogen bonding with the nicotinamide carboxamide group show consistent differences in 1H and 15N chemical shift between NADPH and NADP+ in a variety of ternary complexes. B3LYP calculations in model systems of protein-coenzyme interactions show differences in the H-bond geometry and differences in charge distribution between the oxidised and reduced forms of the nicotinamide ring. GIAO isotropic nuclear shieldings calculated for nuclei in these systems reproduce the experimentally observed trends in magnitudes and signs of the chemical shifts. The experimentally observed reduction in binding of NADP+ compared with NADPH results partly from NADP+ having to change its nicotinamide amide group from a cis- to a trans-conformation on binding and partly from the oxidised nicotinamide ring of NADP+ being unable to take up its optimal hydrogen bonding geometry in its interactions with protein residues.
Full-text · Article · Jan 2002 · Journal of Molecular Structure