Johan Desmet

Conformational splitting: A more powerful criterion for dead‐end elimination

Dead‐end elimination (DEE) is a powerful theorem for selecting optimal protein side‐chain orientations from a large set of discrete conformations. The present work describes a new approach to dead‐end elimination that effectively splits conformational space into partitions to more efficiently eliminate dead‐ending rotamers. Split DEE makes it possible to complete protein design calculations that were previously intractable due to the combinatorial explosion of intermediate conformations generated during the convergence process.

All in one: A highly detailed rotamer library improves both accuracy and speed in the modelling of sidechains by dead-end elimination

BACKGROUND About a decade ago, the concept of rotamer libraries was introduced to model sidechains given known mainchain coordinates. Since then, several groups have developed methods to handle the challenging combinatorial problem that is faced when searching rotamer libraries. To avoid a combinatorial explosion, the dead-end elimination method detects and eliminates rotamers that cannot be members of the global minimum energy conformation (GMEC). Several groups have applied and further developed this method in the fields of homology modelling and protein design. RESULTS This work addresses at the same time increased prediction accuracy and calculation speed improvements. The proposed enhancements allow the elimination of more than one-third of the possible rotameric states before applying the dead-end elimination method. This is achieved by using a highly detailed rotamer library allowing the safe application of an energy-based rejection criterion without risking the elimination of a GMEC rotamer. As a result, we gain both in modelling accuracy and in computational speed. Being completely automated, the current implementation of the dead-end elimination prediction of protein sidechains can be applied to the modelling of sidechains of proteins of any size on the high-end computer systems currently used in molecular modelling. The improved accuracy is highlighted in a comparative study on a collection of proteins of varying size for which score results have previously been published by multiple groups. Furthermore, we propose a new validation method for the scoring of the modelled structure versus the experimental data based upon the volume overlap of the predicted and observed sidechains. This overlap criterion is discussed in relation to the classic RMSD and the frequently used +/- 40 degrees window in comparing chi 1 and chi 2 angles. CONCLUSIONS We have shown that a very detailed library allows the introduction of a safe energy threshold rejection criterion, thereby increasing both the execution speed and the accuracy of the modelling program. We speculate that the current method will allow the sidechain prediction of medium-sized proteins and complex protein interfaces involving up to 150 residues on low-end desktop computers.

Fast and accurate side‐chain topology and energy refinement (FASTER) as a new method for protein structure optimization

We have developed an original method for global optimization of protein side‐chain conformations, called the Fast and Accurate Side‐Chain Topology and Energy Refinement (FASTER) method. The method operates by systematically overcoming local minima of increasing order. Comparison of the FASTER results with those of the dead‐end elimination (DEE) algorithm showed that both methods produce nearly identical results, but the FASTER algorithm is 100–1000 times faster than the DEE method and scales in a stable and favorable way as a function of protein size. We also show that low‐order local minima may be almost as accurate as the global minimum when evaluated against experimentally determined structures. In addition, the new algorithm provides significant information about the conformational flexibility of individual side‐chains. We observed that strictly rigid side‐chains are concentrated mainly in the core of the protein, whereas highly flexible side‐chains are found almost exclusively among solvent‐oriented residues. Proteins 2002;48:31–43.

Development and application of cytotoxic T lymphocyte‐associated antigen 4 as a protein scaffold for the generation of novel binding ligands

We have explored the possibilities of using human cytotoxic T lymphocyte‐associated antigen 4 (CTLA‐4) as a single immunoglobulin fold‐based scaffold for the generation of novel binding ligands. To obtain a suitable protein library selection system, the extracellular domain of CTLA‐4 was first displayed on the surface of a filamentous phage as a fusion product of the phage coat protein p3. CTLA‐4 was shown to be functionally intact by binding to its natural ligands B7‐1 (CD80) and B7‐2 (CD86) both in vitro and in situ. Secondly, the complementarity determining region 3 (CDR3) loop of the CTLA‐4 extracellular domain was evaluated as a permissive site. We replaced the nine amino acid CDR3‐like loop of CTLA‐4 with the sequence XXX‐RGD‐XXX (where X represents any amino acid). Using phage display we selected several CTLA‐4‐based variants capable of binding to human αvβ3 integrin, one of which showed binding to integrins in situ. To explore the construction of bispecific molecules we also evaluated one other potential permissive site diametrically opposite the natural CDR‐like loops, which was found to be tolerant of peptide insertion. Our data suggest that CTLA‐4 is a suitable human scaffold for engineering single‐domain molecules with one or possibly more binding specificities.

Computation of the binding of fully flexible peptides to proteins with flexible side chains.

Docking algorithms play an important role in the process of rational drug design and in understanding the mechanism of molecular recognition. An important determinant for successful docking is the extent to which the configurational space (including conformational changes) of the li‐gand/receptor system is searched. Here we describe a new, combinatorial method for flexible docking of peptides to proteins that allows full rotation around all single bonds of the peptide ligand and around those of a large set of receptor side chains. We have simulated the binding of several viral peptides to murine major histocompatibility complex class I H‐2Kb. In addition, we have explored the limits of our method by simulating a complex between calmodulin and an 18‐residue long helical peptide from calmodulin‐dependent protein kinase IIα. The calculated peptide conformations generally matched well with the X‐ray structures. Essential information about local flexibility and about residues that are responsible for strong binding was obtained. We have frequently observed considerable side‐chain flexibility during the simulations, showing the need for a flexible treatment of the receptor. Our method may also be useful whenever the receptor side‐chain conformation is not available or uncertain, as illustrated by the docking of an H‐2Kb binding nonapeptide to the receptor structure taken from an octapeptide/H‐2Kb complex.—Desmet, J., Wilson, I. A., Joniau, M., De Maeyer, M., Lasters, I. Computation of the binding of fully flexible peptides to proteins with flexible side chains. FASEB J 11, 164‐172 (1997)

Anchor profiles of HLA-specific peptides: Analysis by a novel affinity scoring method and experimental validation

The study of intermolecular interactions is a fundamental research subject in biology. Here we report on the development of a quantitative structure‐based affinity scoring method for peptide–protein complexes, named PepScope. The method operates on the basis of a highly specific force field function (CHARMM) that is applied to all‐atom structural representations of peptide–receptor complexes. Peptide side‐chain contributions to total affinity are scored after detailed rotameric sampling followed by controlled energy refinement. A de novo approach to estimate dehydration energies was developed, based on the simulation of individual amino acids in a solvent box filled with explicit water molecules. Transferability of the method was demonstrated by its application to the hydrophobic HLA‐A2 and ‐A24 receptors, the polar HLA‐A1, and the sterically ruled HLA‐B7 receptor. A combined theoretical and experimental study on 39 anchor substitutions in FxSKQYMTx/HLA‐A2 and ‐A24 complexes indicated a prediction accuracy of about two thirds of a log‐unit in Kd. Analysis of free energy contributions identified a great role of desolvation and conformational strain effects in establishing a given specificity profile. Interestingly, the method rightly predicted that most anchor profiles are less specific than so far assumed. This suggests that many potential T‐cell epitopes could be missed with current prediction methods. The results presented in this work may therefore significantly affect T‐cell epitope discovery programs applied in the field of peptide vaccine development. Proteins 2005.

Rational humanization of the powerful antithrombotic anti-GPIbα antibody: 6B4

Fab-fragments of the monoclonal antibody 6B4, raised against human glycoprotein Ibα (GPIbα), have a powerful antithrombotic effect in baboons by blocking the GPIbα binding site for von Willebrand factor (VWF), without significant prolongation of the skin bleeding time. In order to bring this antibody to the clinic,we here humanized for the first time an anti-human GPIbα by variable-domain resurfacing guided by computer modeling. First, the genes coding for the variable regions of the heavy and light chains of 6B4 were cloned and sequenced. Based on this, a three-dimensional structure of the Fv-fragment was constructed by using homology-based modeling, and with this and comparison with antibodies with known structure,“murine” putative immunogenic residues which are exposed, were changed for “human-like” residues. The humanized Fab-fragment, h6B4-Fab, was constructed in the pKaneo vector system, expressed and purified and showed in vitro an unaltered, even slightly higher binding affinity for its antigen than the murine form as determined by different ELISA set-ups and surface plasmon resonance. Finally, injection of doses of 0.1 to 1.5 mg/kg of h6B4-Fab in baboons showed that both pharmacokinetics and ex-vivo bio-activity of the molecule were to a large extent preserved. In conclusion,the method used here to humanize 6B4 by resurfacing resulted in a fully active derivative, which is now ready for further development.

Structural basis of IL-23 antagonism by an Alphabody protein scaffold

Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein–protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.

Comparison of the binding of Ca2+ and Mn2+ to bovine α-lactalbumin and equine lysozyme

Abstract The enthalpy change of the binding of Ca 2+ and Mn 2+ to equine lysozyme was measured at 25°C and pH 7.5 by batch microcalorimetry: ΔH° Ca 2+ = −76 ± 5 kJ mol −1 , ΔH° Mn 2+ = −21 ± 10 kJ mol −1 . Binding constants, log K Ca 2+ = 6.5 ± 0.2 and log K Mn 2+ = 4.1 ± 0.5, were calculated from the calorimetric data. Therefore, ΔS Ca2+ ° = −131 ± 20 JK −1 mol −1 and ΔS° Mn 2+ = 8 ± 44 JK −1 mol −1 . Removal of Ca 2+ induces small but significant changes in the circular dichroism spectrum, indicating the existence of a partially unfolded apo-conformation, comparable with, but different from, the apo-conformation of bovine α-lactalbumin.

Coexistence of hypocalciuric hypercalcaemia and interstitial lung disease in a family: a cross-sectional study

Abstract. In a prospective investigation, a large kindred (twenty‐one subjects) with unexplained association of familial hypocalciuric hypercalcaemia and idiopathic interstitial lung disease was studied. Serum calcium was increased in fifteen patients (the youngest being 7 years old) and was associated with hypo‐ or normocalciuria. The abnormalities were not age‐dependent. The serum concentrations of parathyroid hormone, 25–hydroxyvitamin D3, 1,25‐dihydroxyvitamin D3 and calcitonin were normal. In twelve patients the diffusing capacity (DLco) and/or DLco per unit lung volume was less than 75% predicted. This was often accompanied by a vital capacity of less than 80% predicted, an increased Tiffeneau index, and a reticulo‐micronodular pattern with high diaphragm on chest X‐ray. The decrease in DLco was more pronounced in older non‐smoking as well as smoking subjects (P < 0·02) suggesting a progressing interstitial disease with age. The fibrosing alveolitis, which had been confirmed by open lung biopsy in three subjects, could not be attributed to sarcoïdosis, collagen‐vascular disease, or exogenous causes. The disturbances in the calcium homeostasis and in the diffusing capacity of the lung coexisted in seven of the twenty‐one patients. Apparently, both abnormalities were inherited following an autosomal‐dominant pattern but with a different penetration in each person, and seemed not be causally related to each other.