Thermal stability of single‐domain antibodies estimated by molecular dynamics simulations

Single‐domain antibodies (sdAbs) function like regular antibodies, however, consist of only one domain. Because of their low molecular weight, sdAbs have advantages with respect to production and delivery to their targets and for applications such as antibody drugs and biosensors. Thus, sdAbs with high thermal stability are required. In this work, we chose seven sdAbs, which have a wide range of melting temperature (T_m) values and known structures. We applied molecular dynamics (MD) simulations to estimate their relative stability and compared them with the experimental data. High‐temperature MD simulations at 400 K and 500 K were executed with simulations at 300 K as a control. The fraction of native atomic contacts, Q, measured for the 400 K simulations showed a fairly good correlation with the T_m values. Interestingly, when the residues were classified by their hydrophobicity and size, the Q values of hydrophilic residues exhibited an even better correlation, suggesting that stabilization is correlated with favorable interactions of hydrophilic residues. Measuring the Q value on a per‐residue level enabled us to identify residues that contribute significantly to the instability and thus demonstrating how our analysis can be used in a mutant case study.     

Molecular dynamics simulations of Alzheimer's beta-amyloid protofilaments

Filamentous amyloid aggregates are central to the pathology of Alzheimer's disease. We use all-atom molecular dynamics (MD) simulations with explicit solvent and multiple force fields to probe the structural stability and the conformational dynamics of several models of Alzheimer's beta-amyloid fibril structures, for both wild-type and mutated amino acid sequences. The structural models are based on recent solid state NMR data. In these models, the peptides form in-register parallel beta-sheets along the fibril axis, with dimers of two U-shaped peptides located in layers normal to the fibril axis. Four different topologies are explored for stacking the beta-strand regions against each other to form a hydrophobic core. Our MD results suggest that all four NMR-based models are structurally stable, and we find good agreement with dihedral angles estimated from solid-state NMR experiments. Asp23 and Lys28 form buried salt-bridges, resulting in an alternating arrangement of the negatively and positively charged residues along the fibril axis that is reminiscent of a one-dimensional ionic crystal. Interior water molecules are solvating the buried salt-bridges. Based on data from NMR measurements and MD simulations of short amyloid fibrils, we constructed structural models of long fibrils. Calculated X-ray fiber diffraction patterns show the characteristics of packed beta-sheets seen in experiments, and suggest new experiments that could discriminate between various fibril topologies.     

A comparative analysis of the equilibrium dynamics of a designed protein inferred from NMR,X‐ray,and computations

A detailed analysis of high‐resolution structural data and computationally predicted dynamics was carried out for a designed sugar‐binding protein. The mean‐square deviations in the positions of residues derived from nuclear magnetic resonance (NMR) models and those inferred from X‐ray crystallographic B‐factors for two different crystal forms were compared with the predictions based on the Gaussian Network Model (GNM) and the results from molecular dynamics (MD) simulations. GNM systematically yielded a higher correlation than MD, with experimental data, suggesting that the lack of atomistic details in the coarse‐grained GNM is more than compensated for by the mathematically exact evaluation of fluctuations using the native contacts topology. Evidence is provided that particular loop motions are curtailed by intermolecular contacts in the crystal environment causing a discrepancy between theory and experiments. Interestingly, the information conveyed by X‐ray crystallography becomes more consistent with NMR models and computational predictions when ensembles of X‐ray models are considered. Less precise (broadly distributed) ensembles indeed appear to describe the accessible conformational space under native state conditions better than B‐factors. Our results highlight the importance of using multiple conformations obtained by alternative experimental methods, and analyzing results from both coarse‐grained models and atomic simulations, for accurate assessment of motions accessible to proteins under native state conditions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Computational modelling and molecular dynamics simulations of a cyclic peptide mimotope of the CD52 antigen complexed with CAMPATH-1H antibody

A peptide mimotope of the CD52 antigen with the sequence T₁SSPSAD₇ has been co-crystallised with the CAMPATH-1H antibody. Molecular dynamics (MD) simulations in explicit water of the T₁SSPSAD₇ peptide in both antibody free and bound states showed that the peptide's β-turn remained stable in the bound state but it was eliminated in the free state. Based on the observation that Thr₁ and Ala₆ residues made close contacts through their side chain, a new peptide mimotope is proposed: (S,S)-C₁SSPSCD₇. Thr₁ and Ala₆ residues have been mutated in Cys residues and a disulphide bond has been imposed. The new analogue has been simulated in both antibody bound and free states with MD in explicit water. It was found that the peptide remained in the stable β-turn conformation, both in complexed and free states. The difference in configurational entropy was estimated to be 0.15 kJ/K/mol. However, despite the structural similarity, the cyclic analogue lost more than 25% of its buried surface area contact with the antibody and a couple of critical hydrogen bond interactions were broken. It is concluded that design of cyclic analogues that mimic the bound conformation of peptides should be carefully performed and conformational ‘freezing’ does not necessarily guarantee better binding.     

Generalized correlation for biomolecular dynamics

Correlated motions in biomolecules are often essential for their function, e.g., allosteric signal transduction or mechanical/thermodynamic energy transport. Because correlated motions in biomolecules remain difficult to access experimentally, molecular dynamics (MD) simulations are particular useful for their analysis. The established method to quantify correlations from MD simulations via calculation of the covariance matrix, however, is restricted to linear correlations and therefore misses part of the correlations in the atomic fluctuations. Herein, we propose a general statistical mechanics approach to detect and quantify any correlated motion from MD trajectories. This generalized correlation measure is contrasted with correlations obtained from covariance matrices for the B1 domain of protein G and T4 lysozyme. The new method successfully quantifies correlations and provides a valuable global overview over the functionally relevant collective motions of lysozyme. In particular, correlated motions of helix 1 together with the two main lobes of lysozyme are detected, which are not seen by the conventional covariance matrix. Overall, the established method misses more than 50% of the correlation. This failure is attributed to both, an interfering and unnecessary dependence on mutual orientations of the atomic fluctuations and, to a lesser extent, attributed to nonlinear correlations. Our generalized correlation measure overcomes these problems and, moreover, allows for an improved understanding of the conformational dynamics by separating linear and nonlinear contributions of the correlation.     

Investigation of glucose binding sites on insulin

Possible insulin binding sites for D-glucose have been investigated theoretically by docking and molecular dynamics (MD) simulations. Two different docking programs for small molecules were used; Multiple Copy Simultaneous Search (MCSS) and Solvation Energy for Exhaustive Docking (SEED) programs. The configurations resulting from the MCSS search were evaluated with a scoring function developed to estimate the binding free energy. SEED calculations were performed using various values for the dielectric constant of the solute. It is found that scores emphasizing non-polar interactions gave a preferential binding site in agreement with that inferred from recent fluorescence and NMR NOESY experiments. The calculated binding affinity of -1.4 to -3.5 kcal/mol is within the measured range of -2.0 +/- 0.5 kcal/mol. The validity of the binding site is suggested by the dynamical stability of the bound glucose when examined with MD simulations with explicit solvent. Alternative binding sites were found in the simulations and their relative stabilities were estimated. The motions of the bound glucose during molecular dynamics simulations are correlated with the motions of the insulin side chains that are in contact with it and with larger scale insulin motions. These results raise the question of whether glucose binding to insulin could play a role in its activity. The results establish the complementarity of molecular dynamics simulations and normal mode analyses with the search for binding sites proposed with small molecule docking programs.     

Reproducible polypeptide folding and structure prediction using molecular dynamics simulations

The folding of a polypeptide from an extended state to a well-defined conformation is studied using microsecond classical molecular dynamics (MD) simulations and replica exchange molecular dynamics (REMD) simulations in explicit solvent and in vacuo. It is shown that the solvated peptide folds many times in the REMD simulations but only a few times in the conventional simulations. From the folding events in the classical simulations we estimate an approximate folding time of 1-2 micros. The REMD simulations allow enough sampling to deduce a detailed Gibbs free energy landscape in three dimensions. The global minimum of the energy landscape corresponds to the native state of the peptide as determined previously by nuclear magnetic resonance (NMR) experiments. Starting from an extended state it takes about 50 ns before the native structure appears in the REMD simulations, about an order of magnitude faster than conventional MD. The calculated melting curve is in good qualitative agreement with experiment. In vacuo, the peptide collapses rapidly to a conformation that is substantially different from the native state in solvent.     

Conformational and dynamics changes induced by bile acids binding to chicken liver bile acid binding protein

The correlation between protein motions and function is a central problem in protein science. Several studies have demonstrated that ligand binding and protein dynamics are strongly correlated in intracellular lipid binding proteins (iLBPs), in which the high degree of flexibility, principally occurring at the level of helix-II, CD, and EF loops (the so-called portal area), is significantly reduced upon ligand binding. We have recently investigated by NMR the dynamic properties of a member of the iLBP family, chicken liver bile acid binding protein (cL-BABP), in its apo and holo form, as a complex with two bile salts molecules. Binding was found to be regulated by a dynamic process and a conformational rearrangement was associated with this event. We report here the results of molecular dynamics (MD) simulations performed on apo and holo cL-BABP with the aim of further characterizing the protein regions involved in motion propagation and of evaluating the main molecular interactions stabilizing bound ligands. Upon binding, the root mean square fluctuation values substantially decrease for CD and EF loops while increase for the helix-loop-helix region, thus indicating that the portal area is the region mostly affected by complex formation. These results nicely correlate with backbone dynamics data derived from NMR experiments. Essential dynamics analysis of the MD trajectories indicates that the major concerted motions involve the three contiguous structural elements of the portal area, which however are dynamically coupled in different ways whether in the presence or in the absence of the ligands. Motions of the EF loop and of the helical region are part of the essential space of both apo and holo-BABP and sample a much wider conformational space in the apo form. Together with NMR results, these data support the view that, in the apo protein, the flexible EF loop visits many conformational states including those typical of the holo state and that the ligand acts stabilizing one of these pre-existing conformations. The present results, in agreement with data reported for other iLBPs, sharpen our knowledge on the binding mechanism for this protein family.     

Relating side-chain mobility in proteins to rotameric transitions: Insights from molecular dynamics simulations and NMR

The dynamic aspect of proteins is fundamental to understanding protein stability and function. One of the goals of NMR studies of side-chain dynamics in proteins is to relate spin relaxation rates to discrete conformational states and the timescales of interconversion between those states. Reported here is a physical analysis of side-chain dynamics that occur on a timescale commensurate with monitoring by ²H spin relaxation within methyl groups. Motivated by observations made from tens-of-nanoseconds long MD simulations on the small protein eglin c in explicit solvent, we propose a simple molecular mechanics-based model for the motions of side-chain methyl groups. By using a Boltzmann distribution within rotamers, and by considering the transitions between different rotamer states, the model semi-quantitatively correlates the population of rotamer states with ‘model-free’ order parameters typically fitted from NMR relaxation experiments. Two easy-to-use, analytical expressions are given for converting S²_axis’ values (order parameter for C–CH₃ bond) into side-chain rotamer populations. These predict that S²_axis’ values below 0.8 result from population of more than one rotameric state. The relations are shown to predict rotameric sampling with reasonable accuracy on the ps–ns timescale for eglin c and are validated for longer timescales on ubiquitin, for which side-chain residual dipolar coupling (RDC) data have been collected.     

How does it really move? Recent progress in the investigation of protein nanosecond dynamics by NMR and simulation

Nuclear magnetic resonance (NMR) spin relaxation experiments currently probe molecular motions on timescales from picoseconds to nanoseconds. The detailed interpretation of these motions in atomic detail benefits from complementarity with the results from molecular dynamics (MD) simulations. In this mini-review, we describe the recent developments in experimental techniques to study the backbone dynamics from ¹⁵N relaxation and side-chain dynamics from ¹³C relaxation, discuss the different analysis approaches from model-free to dynamics detectors, and highlight the many ways that NMR relaxation experiments and MD simulations can be used together to improve the interpretation and gain insights into protein dynamics.     

Accuracy and precision of NMR relaxation experiments and MD simulations for characterizing protein dynamics

Model-free parameters obtained from nuclear magnetic resonance (NMR) relaxation experiments and molecular dynamics (MD) simulations commonly are used to describe the intramolecular dynamical properties of proteins. To assess the relative accuracy and precision of experimental and simulated model-free parameters, three independent data sets derived from backbone ¹⁵N NMR relaxation experiments and two independent data sets derived from MD simulations of Escherichia coli ribonuclease HI are compared. The widths of the distributions of the differences between the order parameters for pairs of NMR data sets are congruent with the uncertainties derived from statistical analyses of individual data sets; thus, current protocols for analyzing NMR data encapsulate random uncertainties appropriately. Large differences in order parameters for certain residues are attributed to systematic differences between samples for intralaboratory comparisons and unknown, possibly magnetic field-dependent, experimental effects for interlaboratory comparisons. The widths of distributions of the differences between the order parameters for two NMR sets are similar to widths of distributions for an NMR and an MD set or for two MD sets. The linear correlations between the order parameters for an MD set and an NMR set are within the range of correlations observed between pairs of NMR sets. These comparisons suggest that the NMR and MD generalized order parameters for the backbone amide N—H bond vectors are of comparable accuracy for residues exhibiting motions on a fast time scale (<100 ps). Large discrepancies between NMR and MD order parameters for certain residues are attributed to the occurrence of “rare” motional events over the simulation trajectories, the disruption of an element of secondary structure in one of the simulations, and lack of consensus among the experimental data sets. Consequently, (easily detectable) severe distortions of local protein structure and infrequent motional events in MD simulations appear to be the most serious artifacts affecting the accuracy and precision, respectively, of MD order parameters relative to NMR values. In addition, MD order parameters for motions on a fast (<100 ps) timescale are more precisely determined than their NMR counterparts, thereby permitting more detailed dynamic characterization of biologically important residues by MD simulation than is sometimes possible by experimental methods. Proteins 28:481–493, 1997. © 1997 Wiley-Liss, Inc.     

Molecular dynamics simulations of bovine rhodopsin: influence of protonation states and different membrane-mimicking environments

G-protein coupled receptors (GPCRs) are a protein family of outstanding pharmaceutical interest. GPCR homology models, based on the crystal structure of bovine rhodopsin, have been shown to be valuable tools in the drug-design process. The initial model is often refined by molecular dynamics (MD) simulations, a procedure that has been recently discussed controversially. We therefore analyzed MD simulations of bovine rhodopsin in order to identify contacts that could serve as constraints in the simulation of homology models. Additionally, the effect of an N-terminal truncation, the nature of the membrane mimic, the influence of varying protonation states of buried residues and the importance of internal water molecules was analyzed. All simulations were carried out using the program-package GROMACS. While N-terminal truncation negatively influenced the overall protein stability, a stable simulation was possible in both solvent environments. As regards the protonation state of titratable sites, the experimental data could be reproduced by the program UHBD (University of Houston Brownian Dynamics), suggesting its application for studying homology models of GPCRs. A high flexibility was observed for internal water molecules at some sites. Finally, interhelical hydrogen-bonding interactions could be derived, which can now serve as constraints in the simulations of GPCR homology models.     

Collective dynamics of EcoRI-DNA complex by elastic network model and molecular dynamics simulations

Anisotropic network model (ANM) is used to analyze the collective motions of restriction enzyme EcoRI in free form and in complex with DNA. For comparison, three independent molecular dynamics (MD) simulations, each of 1.5 ns duration, are also performed for the EcoRI-DNA complex in explicit water. Although high mobility (equilibrium fluctuations) of inner and outer loops that surround the DNA is consistent in both methods and experiments, MD runs sample different conformational subspaces from which reliable collective dynamics cannot be extracted. However, ANM employed on different conformations from MD simulations indicates very similar collective motions. The stems of the inner loops are quite immobile even in the free enzyme and form a large, almost fixed, pocket for DNA binding. As a result, the residues that make specific and non-specific interactions with the DNA exhibit very low fluctuations in the free enzyme. The vibrational entropy difference between the EcoRI complex and free protein + unkinked DNA is positive (favorable), which may partially counteract the unfavorable enthalpy difference of DNA kink formation. Dynamic domains in EcoRI complex and cross-correlations between residue fluctuations indicate possible means of communication between the distal active sites.     

Motional timescale predictions by molecular dynamics simulations: Case study using proline and hydroxyproline sidechain dynamics

We propose a new approach for force field optimizations which aims at reproducing dynamics characteristics using biomolecular MD simulations, in addition to improved prediction of motionally averaged structural properties available from experiment. As the source of experimental data for dynamics fittings, we use ¹³C NMR spin‐lattice relaxation times T₁ of backbone and sidechain carbons, which allow to determine correlation times of both overall molecular and intramolecular motions. For structural fittings, we use motionally averaged experimental values of NMR J couplings. The proline residue and its derivative 4‐hydroxyproline with relatively simple cyclic structure and sidechain dynamics were chosen for the assessment of the new approach in this work. Initially, grid search and simplexed MD simulations identified large number of parameter sets which fit equally well experimental J couplings. Using the Arrhenius‐type relationship between the force constant and the correlation time, the available MD data for a series of parameter sets were analyzed to predict the value of the force constant that best reproduces experimental timescale of the sidechain dynamics. Verification of the new force‐field (termed as AMBER99SB‐ILDNP) against NMR J couplings and correlation times showed consistent and significant improvements compared to the original force field in reproducing both structural and dynamics properties. The results suggest that matching experimental timescales of motions together with motionally averaged characteristics is the valid approach for force field parameter optimization. Such a comprehensive approach is not restricted to cyclic residues and can be extended to other amino acid residues, as well as to the backbone. Proteins 2014; 82:195–215. © 2013 Wiley Periodicals, Inc.     

Exploring the energy landscape of protein folding using replica-exchange and conventional molecular dynamics simulations

Two independent replica-exchange molecular dynamics (REMD) simulations with an explicit water model were performed of the Trp-cage mini-protein. In the first REMD simulation, the replicas started from the native conformation, while in the second they started from a nonnative conformation. Initially, the first simulation yielded results qualitatively similar to those of two previously published REMD simulations: the protein appeared to be over-stabilized, with the predicted melting temperature 50-150K higher than the experimental value of 315K. However, as the first REMD simulation progressed, the protein unfolded at all temperatures. In our second REMD simulation, which starts from a nonnative conformation, there was no evidence of significant folding. Transitions from the unfolded to the folded state did not occur on the timescale of these simulations, despite the expected improvement in sampling of REMD over conventional molecular dynamics (MD) simulations. The combined 1.42 micros of simulation time was insufficient for REMD simulations with different starting structures to converge. Conventional MD simulations at a range of temperatures were also performed. In contrast to REMD, the conventional MD simulations provide an estimate of Tm in good agreement with experiment. Furthermore, the conventional MD is a fraction of the cost of REMD and continuous, realistic pathways of the unfolding process at atomic resolution are obtained.     

Ribosomal RNA Kink-turn Motif—A Flexible Molecular Hinge

Abstract

Ribosomal RNA K-turn motifs are asymmetric internal loops characterized by a sharp bend in the phosphodiester backbone resulting in “V” shaped structures, recurrently observed in ribosomes and showing a high degree of sequence conservation. We have carried out extended explicit solvent molecular dynamics simulations of selected K-turns, in order to investigate their intrinsic structural and dynamical properties. The simulations reveal an unprecedented dynamical flexibility of the K-turns around their X-ray geometries. The K-turns sample, on the nanosecond timescale, different conformational substates. The overall behavior of the simulations suggests that the sampled geometries are essentially isoenergetic and separated by minimal energy barriers. The nanosecond dynamics of isolated K-turns can be qualitatively considered as motion of two rigid helix stems controlled by a very flexible internal loop which then leads to substantial hinge-like motions between the two stems. This internal dynamics of K-turns is strikingly different for example from the bacterial 5S rRNA Loop E motif or BWYV frameshifting pseudoknot which appear to be rigid in the same type of simulations. Bistability and flexibility of K-turns was also suggested by several recent biochemical studies. Although the results of MD simulations should be considered as a qualitative picture of the K-turn dynamics due to force field and sampling limitations, the main advantage of the MD technique is its ability to investigate the region close to K-turn riboso- mal-like geometries. This part of the conformational space is not well characterized by the solution experiments due to large-scale conformational changes seen in the experiments. We suggest that K-turns are well suited to act as flexible structural elements of ribosomal RNA. They can for example be involved in mediation of large-scale motions or they can allow a smooth assembling of the other parts of the ribosome.     

Coevolving residues of (beta/alpha)(8)-barrel proteins play roles in stabilizing active site architecture and coordinating protein dynamics

Indole-3-glycerol phosphate synthase (IGPS) is a representative of (β/α)₈-barrel proteins—the most common enzyme fold in nature. To better understand how the constituent amino-acids work together to define the structure and to facilitate the function, we investigated the evolutionary and dynamical coupling of IGPS residues by combining statistical coupling analysis (SCA) and molecular dynamics (MD) simulations. The coevolving residues identified by the SCA were found to form a network which encloses the active site completely. The MD simulations showed that these coevolving residues are involved in the correlated and anti-correlated motions. The correlated residues are within van der Waals contact and appear to maintain the active site architecture; the anti-correlated residues are mainly distributed on opposite sides of the catalytic cavity and coordinate the motions likely required for the substrate entry and product release. Our findings might have broad implications for proteins with the highly conserved (βα)₈-barrel in assessing the roles of amino-acids that are moderately conserved and not directly involved in the active site of the (β/α)₈-barrel. The results of this study could also provide useful information for further exploring the specific residue motions for the catalysis and protein design based on the (β/α)₈-barrel scaffold.     

Molecular dynamics simulations of Ac-3Aib-Cage-3Aib-NHMe

Solvents play a stabilising role with the more stable conformations obtained in polar solvents than in vacuo. We investigate to what extent the structural propensities of the pentacyclo-undecane (PCU) cage polypeptide chain of the type Ac-3Aib-Cage-3Aib-NHMe are influenced in implicit water and in explicit solvents: methanol (MEOH), dimethyl sulphoxide (DMSO) and TIP3P water. The sampling of the α-helical conformations of the PCU cage polypeptide was investigated using the in-house modified PARM94 force-field parameters. Analysis of 50 ns molecular dynamics (MD) simulations revealed a tendency of the PCU cage polypeptide to assume bent structures, especially in polar solvents. The choice of solvents was designed to relate the simulations to physiological conditions. The individual amino-isobutyric acid residues predominantly sampled the right-handed and left-handed 3₁₀-helical conformations, indicating that the helical conformations are preferred in all four environments (in vacuo, MEOH, water and DMSO). Additionally, the 100 ns replica exchange MD (REMD) simulations of the PCU cage polypeptide in implicit water revealed more conformational variety present than in explicit solvents, and is more consistent with previous theoretical studies on the PCU cage residue. The present theoretical results may help in rationalising experimental results on these PCU cage polypeptides, and definitely show the importance of a dynamical approach for a correct interpretation and prediction of the conformational behaviour of the PCU cage molecules in different environments.     

On the similarity of properties in solution or in the crystalline state: A molecular dynamics study of hen lysozyme

As protein crystals generally possess a high water content, it is assumed that the behaviour of a protein in solution and in crystal environment is very similar. This assumption can be investigated by molecular dynamics (MD) simulation of proteins in the different environments. Two 2ns simulations of hen egg white lysozyme (HEWL) in crystal and solution environment are compared to one another and to experimental data derived from both X-ray and NMR experiments, such as crystallographic B-factors, NOE atom–atom distance bounds, ³J_{H N}-coupling constants, and ¹H-¹⁵N bond vector order parameters. Both MD simulations give very similar results. The crystal simulation reproduces X-ray and NMR data slightly better than the solution simulation.     

Constructing RNA dynamical ensembles by combining MD and motionally decoupled NMR RDCs: new insights into RNA dynamics and adaptive ligand recognition

We describe a strategy for constructing atomic resolution dynamical ensembles of RNA molecules, spanning up to millisecond timescales, that combines molecular dynamics (MD) simulations with NMR residual dipolar couplings (RDC) measured in elongated RNA. The ensembles are generated via a Monte Carlo procedure by selecting snap-shot from an MD trajectory that reproduce experimentally measured RDCs. Using this approach, we construct ensembles for two variants of the transactivation response element (TAR) containing three (HIV-1) and two (HIV-2) nucleotide bulges. The HIV-1 TAR ensemble reveals significant mobility in bulge residues C24 and U25 and to a lesser extent U23 and neighboring helical residue A22 that give rise to large amplitude spatially correlated twisting and bending helical motions. Omission of bulge residue C24 in HIV-2 TAR leads to a significant reduction in both the local mobility in and around the bulge and amplitude of inter-helical bending motions. In contrast, twisting motions of the helices remain comparable in amplitude to HIV-1 TAR and spatial correlations between them increase significantly. Comparison of the HIV-1 TAR dynamical ensemble and ligand bound TAR conformations reveals that several features of the binding pocket and global conformation are dynamically preformed, providing support for adaptive recognition via a ‘conformational selection’ type mechanism.