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Robert Kaptein

Publications and source records attributed to Robert Kaptein.

29 records · Page 2Linked to original sources
PubMed2004

Structure and flexibility adaptation in nonspecific and specific protein-DNA complexes.

Interaction of regulatory DNA binding proteins with their target sites is usually preceded by binding to nonspecific DNA. This speeds up the search for the target site by several orders of magnitude. We report the solution structure and dynamics of the complex of a dimeric lac repressor DNA binding domain with nonspecific DNA. The same set of residues can switch roles from a purely electrostatic interaction with the DNA backbone in the nonspecific complex to a highly specific binding mode with the base pairs of the cognate operator sequence. The protein-DNA interface of the nonspecific complex is flexible on biologically relevant time scales that may assist in the rapid and efficient finding of the target site.

Bacterial Proteins↗
PubMed2004

An alternate conformation of the hyperthermostable HU protein from Thermotoga maritima has unexpectedly high flexibility.

The homodimeric HU protein from the hyperthermophile Thermotoga maritima (HUTmar) is a model system which can yield insights into the molecular determinants of thermostability in proteins. Unusually for a thermostable protein, HUTmar exists in a structurally heterogeneous state as evidenced by the assignment of two distinct and approximately equally populated forms in solution. Relaxation measurements combined with chemical shift, hydrogen exchange, and nuclear Overhauser enhancement data confirm the main structural features of both forms. In addition, these data support a two-state model for HUTmar in which the major form closely resembles the X-ray structure while the very flexible minor form is less structured. HUTmar may therefore be a new example of the small class of hyperthermostable proteins with unexpected flexibility.

Amino Acid Sequence↗
PubMed2003

Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins.

Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.

Acetylgalactosamine↗
PubMed2003

Molecular dynamics studies of a molecular switch in the glucocorticoid receptor.

The glucocorticoid receptor (GR) is a hormone dependent nuclear receptor that regulates gene transcription when bound to the glucocorticoid response element (GRE). The GRE acts as an allosteric effector, inducing a structural change in the GR DNA-binding domain (GR DBD) upon binding, thereby switching the GR to an active conformation. A similar conformational change can be induced by two single point mutations: Ser459Ala and Pro493Arg. Structural and dynamical aspects of the conformational switch have been investigated by molecular dynamics simulations. Our results indicate that these two mutants, which share a similar phenotype, exert their action at a structural level through different mechanisms. In the Arg(493) mutant, the D-loop and the second helix are stabilized in a conformation that preforms the protein-protein dimer interface. In the Ala(459) mutant, the structurally important hydrogen bond between Arg(496) and Ser(459) is missing, which leads to a core reorganization and a reorientation of the second helical region. Although remote, both in sequence and three dimensional structure, these two mutations induce structural changes that are ultimately reflected in similar regions of the GR DBD structure, namely the D-loop and the short second helical region. These correspond to hot area of the GR DBD that are important both for DNA-binding and for the proper formation of the protein-protein interface. The conformational rearrangements in these area are proposed to decrease unfavorable protein-DNA and protein-protein contacts and allow unspecific DNA-binding leading to the squelching phenotype of the mutants. The GR DBD can thus exist in two states, a transcriptionally active and an inactive state. Switching between these states can be accomplished either by GRE binding or by the described mutations.

Alanine↗
PubMed2003

Use of very long-distance NOEs in a fully deuterated protein: an approach for rapid protein fold determination.

The high sensitivity of modern NMR instrumentation, in combination with full deuteration, enabled the measurement of long-range NOEs between amide protons in a fully deuterated protein corresponding to distances up to 8A. These are beyond the limit normally observed in protonated samples. Such long-distance NOEs could be observed using long mixing times, which became possible due to reduced spin diffusion and T1 relaxation of the amide protons in the fully deuterated sample. This information was used in combination with secondary structure restraints derived from secondary chemical shifts for structure calculations. With these backbone amide proton NOEs only, a unique fold could be obtained with positional root mean square deviations from the average of 1.30 and 2.25 A for backbone and heavy atoms, respectively. Despite the low density of restraints, no mirror image problems were observed. Addition of sidechain NOE information increased the precision of the ensemble and in particular of the core packing. The structures obtained in this way were close to the published crystal structure. NOE completeness analysis revealed that the cumulative completeness is still more than 80% for an 8.0 A cut-off distance.

Amides↗
PubMed2002

X-ray absorption spectroscopic studies of zinc in the N-terminal domain of HIV-2 integrase and model compounds.

X-ray absorption spectroscopy (XAS), including extended X-ray absorption fine structure (EXAFS) and X-ray absorption near-edge structure (XANES) analysis, has been carried out at the Zn K edge of the N-terminal part of the integrase protein of the human immunodeficiency virus, type 2 (HIV-2), and of some zinc coordination compounds. In the presence of excess beta-mercaptoethanol, which was present in the NMR structure elucidation of the protein [Eijkelenboom et al. (1997), Curr. Biol. 7, 739-746; (2000), J. Biomol. NMR, 18, 119-28], the protein spectrum was nearly identical to that recorded in its absence. Comparison of the XANES of the protein with that of model compounds and literature data permits the conclusion that the Zn ion is four-coordinated. The major shell of the EXAFS provides evidence for a mixed (N or O as well as S) coordination sphere, while the minor shells indicate imidazole coordination. Our approach to the analysis of the EXAFS, including quantification of the imidazole by multiple scattering simulations with EXCURV92, was validated on the model compounds. An important result is that with multiple scattering simulations using restraints on the parameters of the imidazole rings the number of imidazoles and their orientation could be determined. The integrase spectra can be fitted with two sulfur ligands at 2.26 A (Debye-Waller-type factor 0.009 A(2)) and two imidazole ligands with the N atoms at 1.99 A (Debye-Waller-type factor 0.005 A(2)). The XAS-derived geometry is fully consistent with that found in the NMR structure determination and, allowing for the Volume contraction due to the temperature difference between the experiments, justifies the restraints applied in the structure calculation (Zn-S and Zn-N distances of 2.3 A and 2.0 A, respectively).

Absorptiometry, Photon↗
PubMed2002

Analysis of protein-carbohydrate interaction at the lower size limit of the protein part (15-mer peptide) by NMR spectroscopy, electrospray ionization mass spectrometry, and molecular modeling.

Structural analysis of minimally sized lectins will offer insights into fundamentals of intermolecular recognition and potential for biomedical applications. We thus moved significantly beyond the natural limit of lectin size to determine the structure of synthetic mini-lectins in solution, their carbohydrate selectivity and the impact of ligand binding on their conformational behavior. Using three disaccharide (Thomsen-Friedenreich antigen; Gal beta 1,3GalNAc alpha 1,R)-binding pentadecapeptides without internal disulfide bridges as role models, we successfully tested a combined strategy with different techniques of NMR spectroscopy, electrospray ionization mass spectrometry, and molecular modeling. In solution, the peptides invariably displayed flexibility with rather limited restrictions, shown by NMR experiments including nearly complete resonance assignments and molecular dynamics simulations. The occurrence of aromatic/nonpolar amino acids in the sequence did not lead to formation of a hydrophobic core known from microbial chitinase modules. Selectivity of disaccharide binding was independently observed by mass spectrometry and NMR analysis. Specific ligand interaction yielded characteristic NMR signal alterations but failed to reduce conformational flexibility significantly. We have thereby proven effectiveness of our approach to analyze even low-affinity interactions (not restricted to carbohydrates as ligands). It will be useful to evaluate the impact of rational manipulation of lead peptide sequences.

Amino Acid Sequence↗
PubMed2002

Mapping the targeted membrane pore formation mechanism by solution NMR: the nisin Z and lipid II interaction in SDS micelles.

Nisin is an example of type-A lantibiotics that contain cyclic lanthionine rings and unusual dehydrated amino acids. Among the numerous pore-forming antimicrobial peptides, type-A lantibiotics form an unique family of post-translationally modified peptides. Via the recognition of cell wall precursor lipid II, nisin has the capacity to form pores against Gram-positive bacteria with an extremely high activity in the nanomolar (nM) range. Here we report a high-resolution NMR spectroscopy study of nisin/lipid II interactions in SDS micelles as a model membrane system in order to elucidate the mechanism of molecular recognition at residue level. The binding to lipid II was studied through (15)N-(1)H HSQC titration, backbone amide proton temperature coefficient analysis, and heteronuclear (15)N[(1)H]-NOE relaxation dynamics experiments. Upon the addition of lipid II, significant changes were monitored in the N-terminal part of nisin. An extremely low amide proton temperature coefficient (Delta delta/Delta T) was found for the amide proton of Ala3 (> -0.1 ppb/K) in the complex form. This suggests tight hydrogen bonding and/or isolation from the bulk solvent for this residue. Large chemical shift perturbations were also observed in the first two rings. In contrast, the C-terminal part of nisin was almost unaffected. This part of the molecule remains flexible and solvent-exposed. On the basis of our results, a multistep pore-forming mechanism is proposed. The N-terminal part of nisin first binds to lipid II, and a subsequent structural rearrangement takes place. The C-terminal part of nisin is possibly responsible for the activation of the pore formation. In light of the emerging antibiotic resistance problems, an understanding of the specific recognition mechanism of nisin with lipid II at the residue specific level may therefore aid in the development of novel antibiotics.

Amides↗
PubMed2002

Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.

The lac repressor-operator system is a model system for understanding protein-DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the specific recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the first high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator 01. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48 degrees of the right lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor-operator system is unique among other protein-DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.

Bacterial Proteins↗
PubMed2002

A residue-specific view of the association and dissociation pathway in protein--DNA recognition.

Signal propagation in biological systems occurs through a series of inter- and intramolecular events, the precise pathways of which remain elusive in most cases. With respect to protein--DNA interactions in particular, little is known about the association and dissociation reaction pathways. Here we show that the exchange of amide protons detected by NMR can be used to characterize, at residue level, the mechanism, kinetics and thermodynamics of Lac headpiece (HP) interaction with DNA operators. Specific protein--DNA contacts responsible for the direct readout of the sequence are formed and broken at distinct time scales. Unfolding of the hinge helices triggers protein--DNA dissociation by progressive destabilization of distinct structural units, which is facilitated by the low stability of the protein in the uncomplexed state. Upon DNA binding, a dramatic alteration in the dynamics of the protein is observed, which may be used advantageously by the biological system to switch between functional states. Hydrogen-deuterium exchange can provide an unusually detailed view of the interaction mechanism of a protein--DNA complex and the associated energetics of DNA recognition with residue-level specificity.

Allosteric Regulation↗