beta2-microglobulin H31Y variant 3D structure highlights the protein natural propensity towards intermolecular aggregation.

beta2-Microglobulin (beta2m) is the non-covalently bound light chain of the human class I major histocompatibility complex (MHC-I). The natural turnover of MHC-I gives rise to the release of beta2m into plasmatic fluids and to its catabolism in the kidney. beta2m dissociation from the heavy chain of the complex is a severe complication in patients receiving prolonged hemodialysis. As a consequence of renal failure, the increasing beta2m concentrations can lead to deposition of the protein as amyloid fibrils. Here we characterize the His31-->Tyr human beta2m mutant, a non-natural form of beta2m that is more stable than the wild-type protein, displaying a ten-fold acceleration of the slow phase of folding. We report the 2.9A resolution crystal structure and the NMR characterization of the mutant beta2m, focussing on selected structural features and on the molecular packing observed in the crystals. Juxtaposition of the four mutant beta2m molecules contained in the crystal asymmetric unit, and specific hydrogen bonds, stabilize a compact protein assembly. Conformational heterogeneity of the four independent molecules, some of their mutual interactions and partial unpairing of the N-terminal beta-strand in one protomer are in keeping with the amyloidogenic properties displayed by the mutant beta2m.

Amyloid fibrils derived from the apolipoprotein A1 Leu174Ser variant contain elements of ordered helical structure.

We recently described a new apolipoprotein A1 variant presenting a Leu174Ser replacement mutation that is associated with a familial form of systemic amyloidosis displaying predominant heart involvement. We have now identified a second unrelated patient with very similar clinical presentation and carrying the identical apolipoprotein A1 mutation. In this new patient the main protein constituent of the amyloid fibrils is the polypeptide derived from the first 93 residues of the protein, the identical fragment to that found in the patient previously described to carry this mutation. The X-ray fiber diffraction pattern obtained from preparations of partially aligned fibrils displays the cross-beta reflections characteristic of all amyloid fibrils. In addition to these cross-beta reflections, other reflections suggest the presence of well-defined coiled-coil helical structure arranged with a defined orientation within the fibrils. In both cases the fibrils contain a trace amount of full-length apolipoprotein A1 with an apparent prevalence of the wild-type species over the variant protein. We have found a ratio of full-length wild-type to mutant protein in plasma HDL of three to one. The polypeptide 1--93 purified from natural fibrils can be solubilized in aqueous solutions containing denaturants, and after removal of denaturants it acquires a monomeric state that, based on CD and NMR studies, has a predominantly random coil structure. The addition of phospholipids to the monomeric form induces the formation of some helical structure, thought most likely to occur at the C-terminal end of the polypeptide.

Molecular mechanics and dynamics of biomolecules using a solvent continuum model.

An easy implementation of molecular mechanics and molecular dynamics simulation using a continuum solvent model is presented that is particularly suitable for biomolecular simulations. The computation of solvation forces is made using the linear Poisson-Boltzmann equation (polar contribution) and the solvent-accessible surface area approach (nonpolar contribution). The feasibility of the methodology is demonstrated on a small protein and a small DNA hairpin. Although the parameters employed in this model must be refined to gain reliability, the performance of the method, with a standard choice of parameters, is comparable with results obtained by explicit water simulations. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1830-1842, 2001

Simulation of electrostatic effects in Fab-antigen complex formation.

A model based on the Poisson-Boltzmann equation has been used to model electrostatics in Anti-p24 (HIV-1) Fab-antigen association. The ionization state at different pH values has been simulated and the results have been used to estimate the stability at different pH values and to generate electrostatic potential maps at physiological ionic strength. The analysis of the electrostatic potential at the solvent-accessible surface shows that residues involved in binding are mostly found in the highest, but also in lowest potential regions. Brownian dynamics simulations have been used to estimate the enhancement of the association rate due to electrostatics which appears limited (approximately 2 at 150 mM ionic strength and approximately 3 at 15 mM ionic strength). A much more pronounced effect is observed upon increase of the charge of the diffusing particle. These results compare well with results obtained previously in similar studies on different systems and may serve to estimate the expected order of magnitude of electrostatic effects on association rates in antibody-antigen systems.

Removal of the N-terminal hexapeptide from human beta2-microglobulin facilitates protein aggregation and fibril formation.

The solution structure and stability of N-terminally truncated beta2-microglobulin (deltaN6beta2-m), the major modification in ex vivo fibrils, have been investigated by a variety of biophysical techniques. The results show that deltaN6beta2-m has a free energy of stabilization that is reduced by 2.5 kcal/mol compared to the intact protein. Hydrogen exchange of a mixture of the truncated and full-length proteins at microM concentrations at pH 6.5 monitored by electrospray mass spectrometry reveals that deltaN6beta2-m is significantly less protected than its wild-type counterpart. Analysis of deltaN6beta2-m by NMR shows that this loss of protection occurs in beta strands I, III, and part of II. At mM concentration gel filtration analysis shows that deltaN6beta2-m forms a series of oligomers, including trimers and tetramers, and NMR analysis indicates that strand V is involved in intermolecular interactions that stabilize this association. The truncated species of beta2-microglobulin was found to have a higher tendency to self-associate than the intact molecule, and unlike wild-type protein, is able to form amyloid fibrils at physiological pH. Limited proteolysis experiments and analysis by mass spectrometry support the conformational modifications identified by NMR and suggest that deltaN6beta2-m could be a key intermediate of a proteolytic pathway of beta2-microglobulin. Overall, the data suggest that removal of the six residues from the N-terminus of beta2-microglobulin has a major effect on the stability of the overall fold. Part of the tertiary structure is preserved substantially by the disulfide bridge between Cys25 and Cys80, but the pairing between beta-strands far removed from this constrain is greatly perturbed.

The solution structure of the C-terminal segment of tau protein.

Pathological changes in the microtubule associated protein tau, leading to tau-containing filamentous lesions, are a major hallmark common to many types of human neurodegenerative diseases, including Alzheimer's disease (AD). No structural data are available which could rationalize the extensive conformational changes that occur when tau protein is converted to Alzheimer's paired helical filaments (PHF). The C-terminal portion of tau plays a crucial role in the aggregation of tau into PHF and in the truncation process that generates cytotoxic segments of tau. Therefore, we investigated the solution structure of the hydrophobic C-terminal segment 423-441 of tau protein (PQLATLADEVSASLAKQGL) by 1H 2D NMR spectroscopy. The peptide displays the typical NMR evidence consistent with a alpha-helix geometry with a stabilizing C-capping motif. The reported data represent the first piece of structural information on an important portion of the molecule and can have implications towards the understanding of its pathophysiology.

Biomolecular electrostatics with the linearized Poisson-Boltzmann equation.

Electrostatics plays a key role in many biological processes. The Poisson-Boltzmann equation (PBE) and its linearized form (LPBE) allow prediction of electrostatic effects for biomolecular systems. The discrepancies between the solutions of the PBE and those of the LPBE are well known for systems with a simple geometry, but much less for biomolecular systems. Results for high charge density systems show that there are limitations to the applicability of the LPBE at low ionic strength and, to a lesser extent, at higher ionic strength. For systems with a simple geometry, the onset of nonlinear effects has been shown to be governed by the ratio of the electric field over the Debye screening constant. This ratio is used in the present work to correct the LPBE results to reproduce fairly accurately those obtained from the PBE for systems with a simple geometry. Since the correction does not involve any geometrical parameter, it can be easily applied to real biomolecular systems. The error on the potential for the LPBE (compared to the PBE) spans few kT/q for the systems studied here and is greatly reduced by the correction. This allows for a more accurate evaluation of the electrostatic free energy of the systems.

Selective NMR Experiments on Macromolecules: Implementation and Analysis of QUIET-NOESY.

The QUIET-NOESY experiment (Zwahlen et al., J. Am. Chem Soc. 116, 362-368, 1994) is applied to measure the mobility of the flexible extensions in the large aggregate (800 kDa) of a small heat-shock protein. The proper choices of the experimental protocol and parameters are discussed in order to employ a simplified data analysis procedure. Further experimental verification of the proposed strategy is also presented using the cyclic peptide gramicidin S as a model compound. Under suitable conditions, the determinations based on the analysis of QUIET-NOESY data are affected to a negligible extent by the approximations that are introduced by the proposed approach. Copyright 1998 Academic Press.

Deuterium distribution in lactate as tracer of metabolic pathways.

2H-NMR spectroscopy has been used to study the distribution of 2H atoms in brain lactate, after intraperitoneal injection of 2H2O. Information on brain metabolism was obtained from the ratio [(3-2H) lactate]/[(2-2H)-lactate] which was found very sensitive to the PO2 in the breathing air. The dependence of this ratio on the rates of metabolic pathways is an example of the possibility to use the distribution of 2H atoms in a molecule to probe metabolic flow through different pathways.

Hydrogen-deuterium exchange studies of the rat thyroid transcription factor 1 homeodomain.

The 1H NMR solution structure of the rat thyroid transcription factor 1 homeodomain (TTF-1 HD) showed that the molecule folds like classical homeodomains. The C-terminal extension of helix III (fragment 51-59) appeared to adopt a helical geometry, albeit not as rigid as the preceding portion, but the hydrogen-deuterium exchange of backbone amides and the NOE data provided evidence of a discontinuity between the two moieties of helix III at the highly conserved fragment Asn51-His52-Arg53. Analysis of quantitative measurements of isotope exchange rates allows one to recognize the general occurrence, in that region of HD motifs, of opposite effects to helix III stability. Asparagine, histidine and arginine residues occur most frequently at the beginning and end of protein helices. In TTF-1 HD a local fluctuation is observed in the fragment 51-53 which either kinks or tightens the alpha-helix. A search through the protein structure database reveals that the three most common variants of HD fragments 51-53 are often involved in helices and, frequently, in helix initiation or termination. For homeodomains in general, the nature of the fragment 51-53 may be related to the conformational dynamics of their DNA-recognition helix (helix III). Besides the specific results on fragment 51-53, the complete isotope exchange analysis of TTF-1 HD data shows that the partially solvent-exposed recognition helix is stabilized by hydrophobic interactions, like most of the structured regions of the molecule. Hydrophobic stabilization of the contacting regions meets the requirements of a DNA-interaction mechanism which, as shown with other DNA-protein complexes, should entail negative heat capacity variations due to changes in solvent exposure of the nonpolar protein surface.

Structural comparison between retro-inverso and parent peptides: molecular basis for the biological activity of a retro-inverso analogue of the immunodominant fragment of VP1 coat protein from foot-and-mouth disease virus.

Antibodies induced against intact foot-and-mouth disease Virus (FMDV) particles bind to the retro-inverso analogue of fragment 141-159 of the viral coat protein VP1 of FMDV, variant A, equally well as to the parent peptide. A conformational investigation of this retro-inverso peptide was carried out by nmr spectroscopy and restrained molecular modeling in order to identify the structural basis for the antigenic mimicry between these retro-inverso and parent peptides. In 100% trifluoroethanol a well-defined left-handed alpha-helical region exists from residue 150 to residue 159, which is consistently present in all conformational families obtained from restrained modelling. A less-defined left-handed helical region is present in the tract 144-148, which is also consistent for all structures. Conformational flexibility exists about Gly149, which leads to two types of structures, either bent or linear. In the bent structures, a three-residue inverse tight turn is found, which can be classified as an inverse gamma-turn centered at Gly149. The overall structural features of the retro-inverso peptide are shown to be similar to those of the parent L-peptide. The two molecules, however, are roughly mirror images because they share inherently chiral secondary structure elements. By comparing these conformational conclusions with the x-ray structure of the Fab complex of a corresponding VP1 antigenic fragment, a rationale is proposed to account for the topological requirements of specific recognition that are implied by the equivalent antigenic activity of the natural and retro-inverso compounds.

Electrostatic effects in homeodomain-DNA interactions.

We report here an investigation of the role of electrostatics in homeodomain-DNA interactions using techniques based around the use of the Poisson-Boltzmann equation. In the present case such a study is of particular interest, since in contrast to other proteins previously studied with this method, the homeodomain is a small, highly charged protein that forms extensive ion pairs upon binding DNA. We have investigated the salt dependence of the binding constant for specific association and for a variety of models for non-specific association. The results indicate that, in line with the models proposed by Manning and Record, the entropy of counterion release accounts for a significant fraction of the salt dependence of the binding free energy, though this is perhaps due to fortuitous cancellation of other contributing terms. The thermodynamic effects of a number of specific homeodomain mutants were also investigated, and partly rationalized in terms of favorable electrostatic interactions in the major goove of DNA. Investigation of the temperature-dependence of the free energy of association indicates that the electrostatic contributions become increasingly favorable as the temperature rises. For this particular system, however, there appears to be no significant electrostatic contribution to the delta(delta C(p)) of association. Finally, an analysis of the free energy of interaction when the homeodomain is moved ca one Debye length from the DNA suggests that pure electrostatic forces are able to steer the homeodomain into a partially correct orientation for binding to the DNA.

Improved processing of selective NMR spectra of biopolymers by separation of noise and signal subspaces through singular-value decomposition.

A novel sensitivity-enhancement procedure is introduced for 2D NMR data matrices. It is based on the separation of the signal-and-noise subspaces by means of singular-value decomposition of the experimental 2D array. Although no theoretical limitation exists for a general application of the method, the reliability of the results increases considerably with reduced data sets such as those of selective measurements. Advantageous applications can be envisaged for the quantitation of NMR parameters in biopolymers whose linewidths are often large enough to undermine severely the sensitivity of selective experiments.

Analysis of the solution structure of the homeodomain of rat thyroid transcription factor 1 by 1H-NMR spectroscopy and restrained molecular mechanics.

The solution structure of the rat thyroid transcription factor 1 (TTF-1) homeodomain has been elucidated by 1H-NMR and restrained modeling. The TTF-1 homeodomain folds in the same manner as classical homeodomains, with three helices, a loose loop between the first two helices, and a tight turn between helix II and helix III. The typical assembly of the hydrophobic core is maintained and N-capping motifs are identified in helix I and helix III. The N-terminal stretch of helix II exhibits some mobility, similar to the preceding loop region, which may be related to its anomalous capping. The N-terminal decapeptide and the C-terminal octapeptide of the molecule (68 residues long) are disordered. All the previous characteristics are shared by all known isolated homeodomain structures. An important difference among these structures occurs at the C-terminal extension of helix III, which is either disordered or helically folded. In the TTF-1 homeodomain, the C-terminal extension of helix III (residues 51-59) appears structured, albeit not as rigidly as the preceding portion. Analysis of the NOEs and hydrogendeuterium exchange of backbone amides provides evidence for discontinuity between the two moieties of helix III, which is introduced by a tightening or a kink of residues 51-53.

A molecular code dictates sequence-specific DNA recognition by homeodomains.

Most homeodomains bind to DNA sequences containing the motif 5'-TAAT-3'. The homeodomain of thyroid transcription factor 1 (TTF-1HD) binds to sequences containing a 5'-CAAG-3' core motif, delineating a new mechanism for differential DNA recognition by homeodomains. We investigated the molecular basis of the DNA binding specificity of TTF-1HD by both structural and functional approaches. As already suggested by the three-dimensional structure of TTF-1HD, the DNA binding specificities of the TTF-1, Antennapedia and Engrailed homeodomains, either wild-type or mutants, indicated that the amino acid residue in position 54 is involved in the recognition of the nucleotide at the 3' end of the core motif 5'-NAAN-3'. The nucleotide at the 5' position of this core sequence is recognized by the amino acids located in position 6, 7 and 8 of the TTF-1 and Antennapedia homeodomains. These data, together with previous suggestions on the role of amino acids in position 50, indicate that the DNA binding specificity of homeodomains can be determined by a combinatorial molecular code. We also show that some specific combinations of the key amino acid residues involved in DNA recognition do not follow a simple, additive rule.

Modeling of polypeptide chains as C alpha chains, C alpha chains with C beta, and C alpha chains with ellipsoidal lateral chains.

In an effort to reduce the number of degrees of freedom necessary to describe a polypeptide chain we analyze the statistical behavior of polypeptide chains when represented as C alpha chains, C alpha chains with C beta atoms attached, and C alpha chains with rotational ellipsoids as models of side chains. A statistical analysis on a restricted data set of 75 unrelated protein structures is performed. The comparison of the database distributions with those obtained by model calculation on very short polypeptide stretches allows the dissection of local versus nonlocal features of the distributions. The database distribution of the bend angles of polypeptide chains of pseudo bonded C alpha atoms spans a restricted range of values and shows a bimodal structure. On the other hand, the torsion angles of the C alpha chain may assume almost all possible values. The distribution is bimodal, but with a much broader probability distribution than for bend angles. The C alpha - C beta vectors may be taken as representative of the orientation of the lateral chain, as the direction of the bond is close to the direction of the vector joining C alpha to the ad hoc defined center of the "steric mass" of the side chain. Interestingly, both the bend angle defined by C alpha i-C alpha i+1-C beta i+1 and the torsional angle offset of the pseudo-dihedral C alpha i-C alpha i+1-C alpha i+2-C beta i+2 with respect to C alpha i-C alpha i+1-C alpha i+2-C alpha i+3 span a limited range of values. The latter results show that it is possible to give a more realistic representation of polypeptide chains without introducing additional degrees of freedom, i.e., by just adding to the C alpha chain a C beta with given side-chain properties. However, a more realistic description of side chains may be attained by modeling side chains as rotational ellipsoids that have roughly the same orientation and steric hindrance. To this end, we define the steric mass of an atom as proportional to its van der Waals volume and we calculate the side-chain inertia ellipsoid assuming that the steric mass of each atom is uniformly distributed within its van der Waals volume. Finally, we define the rotational ellipsoid representing the side chain as the uniform density ellipsoid possessing the same rotationally averaged inertia tensor of the side chain. The statistics of ellipsoid parameters support the possibility of representing a side chain via an ellipsoid, independently of the local conformation. To make this description useful for molecular modeling we describe ellipsoid-ellipsoid interactions via a Lennard-Jones potential that preserves the repulsive core of the interacting ellipsoids and takes into account their mutual orientation. Tests are performed for two different forms of the interaction potential on a set of high-resolution protein structures. Results are encouraging, in view of the drastic simplifications that were introduced.

Structural study of rat thyroid transcription factor 1 homeodomain (TTF-1 HD) by nuclear magnetic resonance.

The 500 MHz 1H NMR spectrum of a 68-residue peptide, encompassing the rat thyroid transcription factor 1 homeodomain (TTF-1 HD), was fully assigned using standard 2D NMR methodology. The secondary structure elements and their spatial organization were determined and led to a structure very similar to that previously described for other homeodomains and expected also for TTF-1 HD from homology modeling predictions. The three-dimensional arrangement of the three helix fragments of TTF-1 HD preserves the helix-turn-helix motif commonly occurring in many classes of DNA-binding proteins.

Free energies calculated according to Manning's polyelectrolyte model compared with Poisson Boltzmann predictions.

The values of the different terms contributing to the free energy of a polyelectrolyte in ionic solution, calculated according to Manning's model are compared with those predicted by the Poisson-Boltzmann(P-B) equation solved for the cell model. On this ground, the limits of applicability of Manning's model and some of its features are discussed. The comparison confirms the usefulness of Manning's model for low charge density polyelectrolytes, while for higher charge densities some care should be used due to the breakdown of the approximations underlying the model.

Conformational study of a short Pertussis toxin T cell epitope incorporated in a multiple antigen peptide template by CD and two-dimensional NMR. Analysis of the structural effects on the activity of synthetic immunogens.

The immunogenic efficacy of multiple antigen peptides, MAPs, i.e. branched molecules in which multiple copies of a given immunogenic peptide are attached on a scaffold of lysine residues via both alpha and epsilon linkages, has been repeatedly demonstrated, but little is known about the structural arrangement of these peptide constructs. A conformational characterization was therefore performed for a known T cell epitope of the S1 subunit of Pertussis toxin, whose sequence is predicted to form alpha-helix. The peptide DNVLDHLTGR, its N-acetylated and C-amidated analogue and a tetrabranched MAP based on the N-acetylated peptide were prepared and studied by CD and two-dimensional 1H-NMR. No evidence of helical structure was obtained in water for the isolated peptides. In contrast, in triflouroethanol, the isolated epitopes fold into a helical structure spanning the segment Val3-Thr8 in the uncapped molecule and encompassing also the N-terminal region in the capped analogue. The mobile C-terminal region tends to adopt a distorted turn arrangement in both peptides due to the folding of Arg10 guanidinium over the backbone. No distortion of the helix structure was observed for the single-copy epitope in the four-branched MAP molecule in trifluoroethanol: each peptide chain is equivalent within the MAP and shows an even more regular helical pattern than the isolated end-blocked sequence. A slight difference was located at the junction with the lysine scaffold: the peptide bond to epsilon NH was found in a much more extended conformation than the corresponding link to alpha NH. These structural results correlate with in vitro T cell stimulatory activity of the three compounds examined and provide arguments supporting the previous suggestion that MAP tetramers are unlikely to elicit an immune response specific for the synthetic template assembly, a feature necessary to retain the advantage of the polymeric epitope presentation.

Homology model building of the thyroid transcription factor 1 homeodomain.

A possible structure for the homeodomain of rat thyroid transcription factor 1 is proposed on the basis of the homology with other homeodomains whose structures have been solved by X-ray crystallography. A structure very similar to the reference ones is feasible and may account for the observed DNA-binding specificity. Structural features of the model, which are likely to be shared by other homeodomains, are discussed and may help researchers in the field to analyse their own experimental data.