On the ability of molecular dynamics force fields to recapitulate NMR derived protein side chain order parameters

Molecular dynamics (MD) simulations have become a central tool for investigating various biophysical questions with atomistic detail. While many different proxies are used to qualify MD force fields, most are based on largely structural parameters such as the root mean square deviation from experimental coordinates or nuclear magnetic resonance (NMR) chemical shifts and residual dipolar couplings. NMR derived Lipari–Szabo squared generalized order parameter (O²) values of amide N? H bond vectors of the polypeptide chain were also often employed for refinement and validation. However, with a few exceptions, side chain methyl symmetry axis order parameters have not been incorporated into experimental reference sets. Using a test set of five diverse proteins, the performance of several force fields implemented in the NAMDD simulation package was examined. It was found that simulations employing explicit water implemented using the TIP3 model generally performed significantly better than those using implicit water in reproducing experimental methyl symmetry axis O² values. Overall the CHARMM27 force field performs nominally better than two implementations of the Amber force field. It appeared that recent quantum mechanics modifications to side chain torsional angles of leucine and isoleucine in the Amber force field have significantly hindered proper motional modeling for these residues. There remained significant room for improvement as even the best correlations of experimental and simulated methyl group Lipari–Szabo generalized order parameters fall below an R² of 0.8.     

Starting‐structure dependence of nanosecond timescale intersubstate transitions and reproducibility of MD‐derived order parameters

Factors affecting the accuracy of molecular dynamics (MD) simulations are investigated by comparing generalized order parameters for backbone NH vectors of the B3 immunoglobulin‐binding domain of streptococcal protein G (GB3) derived from simulations with values obtained from NMR spin relaxation (Yao L, Grishaev A, Cornilescu G, Bax A, J Am Chem Soc 2010;132:4295‐4309.). Choices for many parameters of the simulations, such as buffer volume, water model, or salt concentration, have only minor influences on the resulting order parameters. In contrast, seemingly minor conformational differences in starting structures, such as orientations of sidechain hydroxyl groups, resulting from applying different protonation algorithms to the same structure, have major effects on backbone dynamics. Some, but not all, of these effects are mitigated by increased sampling in simulations. Most discrepancies between simulated and experimental results occur for residues located at the ends of secondary structures and involve large amplitude nanosecond timescale transitions between distinct conformational substates. These transitions result in autocorrelation functions for bond vector reorientation that do not converge when calculated over individual simulation blocks, typically of length similar to the overall rotational diffusion time. A test for convergence before averaging the order parameters from different blocks results in better agreement between order parameters calculated from different sets of simulations and with NMR‐derived order parameters. Thus, MD‐derived order parameters are more strongly affected by transitions between conformational substates than by fluctuations within individual substates themselves, while conformational differences in the starting structures affect the frequency and scale of such transitions. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Conformational studies of a bicyclic,lactam‐constrained parathyroid hormone‐related protein‐derived agonist

The N‐terminal 1–34 segments of both parathyroid hormone (PTH) and parathyroid hormone‐related protein (PTHrP) bind and activate the same membrane receptor in spite of major differences in their amino acid sequence. The hypothesis was made that they share the same bioactive conformation when bound to the receptor. A common structural motif in all bioactive fragments of the hormone in water/trifluoroethanol mixtures or in aqueous solution containing detergent micelles is the presence of two helical segments at the N‐ and C‐termini of the sequence. In order to stabilize the helical structures, we have recently synthesized and studied the PTHrP(1–34) analog [(Lys13–As p¹⁷, Lys26–As p³⁰)]PTHrP(1–34)NH₂, which contains lactam‐constrained Lys‐Asp side chains at positions i, i+4. This very potent agonist exhibits enhanced helix stability with respect to the corresponding linear peptide and also two flexible sites at positions 12 and 19 in 1:1 trifluoroethanol/water. These structural elements have been suggested to play a critical role in bioactivity. In the present work we have extended our conformational studies on the bicyclic lactam‐constrained analog to aqueous solution. By CD, 2D‐NMR and structure calculations we have shown that in water two helical segments are present in the region of the lactam bridges (13–18, and 26–31) with high flexibility around Gly¹² and Arg¹⁹. Thus, the essential structural features observed in the aqueous‐organic medium are maintained in water even if, in this solvent, the overall structure is more flexible. Our findings confirm the stabilizing effect of side‐chain lactam constraints on the α‐helical structure. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Smoluchowski dynamics of the vnd/NK‐2 homeodomain from Drosophila melanogaster: First‐order mode‐coupling approximation

This work is the first in a series devoted to applying mode coupling diffusion theory to the derivation of local dynamics properties of proteins in solution. The first‐order mode‐coupling approximation, or optimized Rouse–Zimm local dynamics (ORZLD), is applied here to derive the rotational dynamics of the bonds and compare the calculated with the experimental nmr ¹⁵N spin–lattice relaxation time behavior of the vnd/NK‐2 homeodomain from Drosophila melanogaster. The starting point for the calculations is the experimental three‐dimensional structure of the homeodomain determined by multidimensional nmr spectroscopy. The results of the computations are compared with experimentally measured ¹⁵N spin–lattice relaxation times T₁, at 34.5 and 60.8 MHz, to check the first‐order approximation. To estimate the relative importance of internal and overall rotation, both rigid and fluctuating dynamic models are examined, with fluctuations evaluated using molecular dynamics (MD) simulations. The correlation times for the fundamental bond vector time correlation function and for the second‐order bond orientational TCF are obtained as a function of the residue number for vnd/NK‐2. The stability of the corresponding local dynamics pattern for the fluctuating structure as a function of the length of the MD trajectory is presented. Diffusive dynamics, which is essentially free of model parameters even at first order in the mode‐coupling diffusion approach, confirm that local dynamics of proteins can be described in terms of rotational diffusion of a fluctuating quasi‐rigid structure. The comparison with the nmr data shows that the first‐order mode coupling diffusion approximation accounts for the correct order of magnitude of the results and of important qualitative aspects of the data sensitive to conformational changes. Indications are obtained from this study to efficiently extend the theory to higher order in the mode‐coupling expansion. These results demonstrate the promise of the mode‐coupling approach, where the local dynamics of proteins is described in terms of rotational diffusion of a fluctuating quasi‐rigid structure, to analyze nmr spin–lattice relaxation behavior. © 1999 John Wiley & Sons, Inc. Biopoly 49: 235–254, 1999     

Optimized labeling of 13CHD2 methyl isotopomers in perdeuterated proteins: Potential advantages for 13C relaxation studies of methyl dynamics of larger proteins

¹³CHD₂ methyl isotopomers are particularly useful to study methyl dynamics in proteins because, as compared with other methyl isotopomers, the ¹³C relaxation mechanism for this isotopomer is straightforward. However, in the case of proteins, where ()² 1, the refocused INEPT pulse sequence does not completely suppress unwanted ¹³CH₃ signals. The presence of weak ¹³CH₃ peaks is usually not a serious problem for smaller proteins because there are relatively few methyl signals and they are sharp; however, signal overlap becomes more common as the size of the protein increases. We overcome this problem by preparing a protein using a 98% D₂O cell culture medium containing 3-¹³C pyruvic acid, 50–60% deuterated at the 3-position, and 4-¹³C 2-ketobutyric acid, 98% and 62% deuterated at the 3- and 4-positions, respectively. This approach significantly reduces the population of the CH₃ isotopomer while optimizing the production of ¹³CHD₂, the isotopomer desired for ¹³C relaxation measurements. In larger proteins where the deuterium T₂ may be too short to measure accurately, we also suggest the alternative measurement of the proton T₂ of the ¹³CH₂D methyl isotopomer, because these protons are well-isolated from other protons in these highly deuterated samples.     

NMR‐based characterization of a refolding intermediate of β2‐microglobulin labeled using a wheat germ cell‐free system

In patients with dialysis‐related amyloidosis, β2‐microglobulin (β2‐m) is a major structural component of amyloid fibrils. It has been suggested that the partial unfolding of β2‐m is a prerequisite to the formation of amyloid fibrils, and that the folding intermediate trapped by the non‐native trans‐Pro32 isomer leads to the formation of amyloid fibrils. Although clarifying the structure of this refolding intermediate by high resolution NMR spectroscopy is important, this has been made difficult by the limited lifetime of the intermediate. Here, we studied the structure of the refolding intermediate using a combination of amino acid selective labeling with wheat germ cell‐free protein synthesis and NMR techniques. The HSQC spectra of β2‐ms labeled selectively at either phenylalanine, leucine, or valine enabled us to monitor the structures of the refolding intermediate. The results suggested that the refolding intermediate has an overall fold and cores similar to the native structure, but contains disordered structures around Pro32. The fluctuation of the β‐sheet regions especially the last half of the βB strand and the first half of the βE strand, both suggested to be important for amyloidogenicity, may transform β2‐m into an amyloidogenic structure.     

Improved side‐chain torsion potentials for the Amber ff99SB protein force field

Recent advances in hardware and software have enabled increasingly long molecular dynamics (MD) simulations of biomolecules, exposing certain limitations in the accuracy of the force fields used for such simulations and spurring efforts to refine these force fields. Recent modifications to the Amber and CHARMM protein force fields, for example, have improved the backbone torsion potentials, remedying deficiencies in earlier versions. Here, we further advance simulation accuracy by improving the amino acid side‐chain torsion potentials of the Amber ff99SB force field. First, we used simulations of model alpha‐helical systems to identify the four residue types whose rotamer distribution differed the most from expectations based on Protein Data Bank statistics. Second, we optimized the side‐chain torsion potentials of these residues to match new, high‐level quantum‐mechanical calculations. Finally, we used microsecond‐timescale MD simulations in explicit solvent to validate the resulting force field against a large set of experimental NMR measurements that directly probe side‐chain conformations. The new force field, which we have termed Amber ff99SB‐ILDN, exhibits considerably better agreement with the NMR data. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effects of calcium binding on the side-chain methyl dynamics of calbindin D9k: a 2H NMR relaxation study

The effects of Ca(2+) binding on the side-chain methyl dynamics of calbindin D(9k) have been characterized by (2)H NMR relaxation rate measurements. Longitudinal, transverse in-phase, quadrupolar order, transverse anti-phase and double quantum relaxation rates are reported for both the apo and Ca(2+)-loaded states of the protein at two magnetic field strengths. The relatively large size of the data set allows for a detailed analysis of the underlying conformational dynamics by spectral density mapping and model-free fitting procedures. The results reveal a correlation between a methyl group's distance from the Ca(2+) binding sites and its conformational dynamics. Several methyl groups segregate into two limiting classes, one proximal and the other distal to the binding sites. Methyl groups in these two classes respond differently to Ca(2+) binding, both in terms of the timescale and amplitude of their fluctuations. Ca(2+) binding elicits a partial immobilization among methyl groups in the proximal class, which is consistent with previous studies of calbindin's backbone dynamics. The distal class, however, exhibits a trend that could not be inferred from the backbone data in that its mobility actually increases with Ca(2+) binding. We have introduced the term polar dynamics to describe this type of organization across the molecule. The trend may represent an important mechanism by which calbindin D(9k) achieves high affinity binding while minimizing the corresponding loss of conformational entropy.     

Dynamics of the SPRY domain-containing SOCS box protein 2: flexibility of key functional loops

The SPRY domain was identified originally as a sequence repeat in the dual-specificity kinase splA and ryanodine receptors and subsequently found in many other distinct proteins, including more than 70 encoded in the human genome. It is a subdomain of the B30.2/SPRY domain and is believed to function as a protein-protein interaction module. Three-dimensional structures of several B30.2/SPRY domain-containing proteins have been reported recently: murine SSB-2 in solution by NMR spectroscopy, a Drosophila SSB (GUSTAVUS), and human PRYSPRY protein by X-ray crystallography. The three structures share a core of two antiparallel beta-sheets for the B30.2/SPRY domain but show differences located mainly at one end of the beta-sandwich. Analysis of SSB-2 residues required for interactions with its intracellular ligands has provided insights into B30.2/SPRY binding specificity and identified loop residues critical for the function of this domain. We have investigated the backbone dynamics of SSB-2 by means of Modelfree analysis of its backbone (15)N relaxation parameters and carried out coarse-grained dynamics simulation of B30.2/SPRY domain-containing proteins using normal mode analysis. Translational self-diffusion coefficients of SSB-2 measured using pulsed field gradient NMR were used to confirm the monomeric state of SSB-2 in solution. These results, together with previously reported amide exchange data, highlight the underlying flexibility of the loop regions of B30.2/SPRY domain-containing proteins that have been shown to be important for protein-protein interactions. The underlying flexibility of certain regions of the B30.2/SPRY domain-containing proteins may also contribute to some apparent structural differences observed between GUSTAVUS or PRYSPRY and SSB-2.     

Improved anticancer potency by head‐to‐tail cyclization of short cationic anticancer peptides containing a lipophilic β2,2‐amino acid

We have recently reported a series of synthetic anticancer heptapeptides (H‐KKWβ^2,2WKK‐NH₂) containing a central achiral and lipophilic β^2,2‐amino acid that display low toxicity against non‐malignant cells and high proteolytic stability. In the present study, we have further investigated the effects of increasing the rigidity and amphipathicity of two of our lead heptapeptides by preparing a series of seven to five residue cyclic peptides containing the two most promising β^2,2‐amino acid derivatives as part of the central lipophilic core. The peptides were tested for anticancer activity against human Burkitt's lymphoma (Ramos cells), haemolytic activity against human red blood cells (RBC) and cytotoxicity against healthy human lung fibroblast cells (MRC‐5). The results demonstrated a considerable increase in anticancer potency following head‐to‐tail peptide cyclization, especially for the shortest derivatives lacking a tryptophan residue. High‐resolution NMR studies and molecular dynamics simulations together with an annexin‐V‐FITC and propidium iodide fluorescent assay showed that the peptides had a membrane disruptive mode of action and that the more potent peptides penetrated deeper into the lipid bilayer. The need for new anticancer drugs with novel modes of action is demanding, and development of short cyclic anticancer peptides with an overall rigidified and amphipathic structure is a promising approach to new anticancer agents. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Characterization of the backbone dynamics of an anti-digoxin antibody VL domain by inverse detected 1H–15N NMR: Comparisons with X-ray data for the Fab

The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody V_L domain has been characterized by measurements of ¹⁵N T₁ and T₂ relaxation times, ¹H–¹⁵N NOE values, and ¹H–²H exchange rates. These data were acquired with 2D inverse detected heteronuclear ¹H–¹⁵N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R₂ (= 1/T₂). All characterized residues display low-amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R₂ exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R₂ exchange contributions. The measured ¹H–²H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the V_L domain, including the three hypervariable loops, undergo dynamic changes upon V_L:V_H association and/ or complexation with digoxin. Overall, the 26–10 V_L domain exhibits relatively low flexibility on the ps–ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley-Liss, Inc.     

Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design.

Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure-based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme-ligand structures, several elastase-inhibitor complexes, the FK506 binding protein, 30 HIV-1 protease-inhibitor complexes, and a number of small and large protein inhibitors. Medium-sized globular proteins typically have 10-20 pockets/cavities. Most often, binding sites are pockets with 1-2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 10(2)-10(3)A3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < or =700 A3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994, Nat Struct Biol 1:55-58). Analysis of elastase-inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et al., 1996, Science 274:1531-1534), indicates that CAST pocket computation may provide a priori identification of target proteins for linked-fragment design. CAST analysis of 30 HIV-1 protease-inhibitor complexes shows that the flexible active site pocket can vary over a range of 853-1,566 A3, and that there are two pockets near or adjoining the active site that may be recruited for ligand design.     

Enantiopure O‐Ethyl Phenylphosphonothioic Acid: A Solvating Agent for the Determination of Enantiomeric Excesses

An improved method, which is highly reproducible, was developed for the enantioseparation of racemic O‐ethyl phenylphosphonothioic acid ( 1a ) with brucine by introducing seeding to a supersaturated solution of the diastereomeric salt mixture. The present method gave both diastereomeric salts in high yields with a diastereomeric ratio of >99.5:0.5 upon choosing the crystallization solvent (MeOH for the ( (R)-1a salt and MeOH/H₂O for the ( (S)-1a salt). The enantiopure acid (R)-1a , (S)-1a showed a good chirality recognition ability for not only strong bases, such as amines and amino alcohols, but also weakly basic alcohols and was applicable as a solvating agent to the ¹H NMR determination of the enantiomeric excess of chiral amines, amino alcohols, and alcohols, including aliphatic substrates. Chirality 26:614–619, 2014. © 2014 Wiley Periodicals, Inc.     

The improvement of the algorithm for order parameter calculation (S2) from molecular dynamics simulation using the correlation motion function

The generalized order parameter, S2, calculated from MD simulation trajectory using time-dependent internal Correlation Motion Function (CMF) agrees well with NMR derived S2 processed with the extended model-free analysis approach. However, the former lies considerably lower comparing to simple model-free derived data from NMR experiments. In the present study we analyze possible reasons of such disagreement. In the general case we propose to use preexponential factors from expression for internal CMF rather than ordinary S2 values. Particularly, in case of the simple model-free S(2) experimental values we suggest comparing them with S2(eff)=1+S2-Sf2 computed from MD simulation data. We show that the S2(eff) values are in a good agreement with NMR derived S2 values obtained using the simple model-free analysis.     

Local sequence of protein β‐strands influences twist and bend angles

β‐Sheet twisting is thought to be mainly determined by interstrand hydrogen bonds with little contribution from side chains, but some proteins have large, flat β‐sheets, suggesting that side chains influence β‐structures. We therefore investigated the relationship between amino acid composition and twists or bends of β‐strands. We calculated and statistically analyzed the twist and bend angles of short frames of β‐strands in known protein structures. The most frequent twist angles were strongly negatively correlated with the proportion of hydrophilic amino acid residues. The majority of hydrophilic residues (except serine and threonine) were found in the edge regions of β‐strands, suggesting that the side chains of these residues likely do not affect β‐strand structure. In contrast, the majority of serine, threonine, and asparagine side‐chains in β‐strands made contacts with a nitrogen atom of the main chain, suggesting that these residues suppress β‐strand twisting. Proteins 2014; 82:1484–1493. © 2014 Wiley Periodicals, Inc.     

Exploring the dynamic information content of a protein NMR structure: Comparison of a molecular dynamics simulation with the NMR and X‐ray structures of Escherichia coli ribonuclease HI

The multiconformer nature of solution nuclear magnetic resonance (NMR) structures of proteins results from the effects of intramolecular dynamics, spin diffusion and an uneven distribution of structural restraints throughout the molecule. A delineation of the former from the latter two contributions is attempted in this work for an ensemble of 15 NMR structures of the protein Escherichia coli ribonuclease HI (RNase HI). Exploration of the dynamic information content of the NMR ensemble is carried out through correlation with data from two crystal structures and a 1.7‐ns molecular dynamics (MD) trajectory of RNase HI in explicit solvent. Assessment of the consistency of the crystal and mean MD structures with nuclear Overhauser effect (NOE) data showed that the NMR ensemble is overall more compatible with the high‐resolution (1.48 Å) crystal structure than with either the lower‐resolution (2.05 Å) crystal structure or the MD simulation. Furthermore, the NMR ensemble is found to span more conformational space than the MD simulation for both the backbone and the sidechains of RNase HI. Nonetheless, the backbone conformational variability of both the NMR ensemble and the simulation is especially consistent with NMR relaxation measurements of two loop regions that are putative sites of substrate recognition. Plausible side‐chain dynamic information is extracted from the NMR ensemble on the basis of (i) rotamericity and syn‐pentane character of variable torsion angles, (ii) comparison of the magnitude of atomic mean‐square fluctuations (msf) with those deduced from crystallographic thermal factors, and (iii) comparison of torsion angle conformational behavior in the NMR ensemble and the simulation. Several heterogeneous torsion angles, while adopting non‐rotameric/syn‐pentane conformations in the NMR ensemble, exist in a unique conformation in the simulation and display low X‐ray thermal factors. These torsions are identified as sites whose variability is likely to be an artifact of the NMR structure determination procedure. A number of other torsions show a close correspondence between the conformations sampled in the NMR and MD ensembles, as well as significant correlations among crystallographic thermal factors and atomic msf calculated from the NMR ensemble and the simulation. These results indicate that a significant amount of dynamic information is contained in the NMR ensemble. The relevance of the present findings for the biological function of RNase HI, protein recognition studies, and previous investigations of the motional content of protein NMR structures are discussed. Proteins 1999;36:87–110. © 1999 Wiley‐Liss, Inc.     

CTD of SARS‐CoV‐2 N protein is a cryptic domain for binding ATP and nucleic acid that interplay in modulating phase separation

SARS‐CoV‐2 nucleocapsid (N) protein plays essential roles in many steps of the viral life cycle, thus representing a key drug target. N protein contains the folded N‐/C‐terminal domains (NTD/CTD) and three intrinsically disordered regions, while its functions including liquid–liquid phase separation (LLPS) depend on the capacity in binding various viral/host‐cell RNA/DNA of diverse sequences. Previously NTD was established to bind various RNA/DNA while CTD to dimerize/oligomerize for forming high‐order structures. By NMR, here for the first time we decrypt that CTD is not only capable of binding S2m, a specific probe derived from SARS‐CoV‐2 gRNA but with the affinity even higher than that of NTD. Very unexpectedly, ATP, the universal energy currency for all living cells with high cellular concentrations (2–16 mM), specifically binds CTD with Kd of 1.49 ± 0.28 mM. Strikingly, the ATP‐binding residues of NTD/CTD are identical in the SARS‐CoV‐2 variants while ATP and S2m interplay in binding NTD/CTD, as well as in modulating LLPS critical for the viral life cycle. Results together not only define CTD as a novel binding domain for ATP and nucleic acid, but enforce our previous proposal that ATP has been evolutionarily exploited by SARS‐CoV‐2 to complete its life cycle in the host cell. Most importantly, the unique ATP‐binding pockets on NTD/CTD may offer promising targets for design of specific anti‐SARS‐CoV‐2 molecules to fight the pandemic. Fundamentally, ATP emerges to act at mM as a cellular factor to control the interface between the host cell and virus lacking the ability to generate ATP.     

Structural insight for the roles of fas death domain binding to fadd and oligomerization degree of the fas–fadd complex in the death‐inducing signaling complex formation: A computational study

Fas binding to Fas‐associated death domain (FADD) activates FADD–caspase‐8 binding to form death‐inducing signaling complex (DISC) that triggers apoptosis. The Fas–Fas association exists primarily as dimer in the Fas–FADD complex, and the Fas–FADD tetramer complexes have the tendency to form higher order oligomer. The importance of the oligomerized Fas–FADD complex in DISC formation has been confirmed. This study sought to provide structural insight for the roles of Fas death domain (Fas DD) binding to FADD and the oligomerization of Fas DD–FADD complex in activating FADD–procaspase‐8 binding. Results show Fas DD binding to FADD stabilized the FADD conformation, including the increased stability of the critical residues in FADD death effector domain (FADD DED) for FADD–procaspase‐8 binding. Fas DD binding to FADD resulted in the decreased degree of both correlated and anticorrelated motion of the residues in FADD and caused the reversed correlated motion between FADD DED and FADD death domain (FADD DD). The exposure of procaspase‐8 binding residues in FADD that allows FADD to interact with procaspase‐8 was observed with Fas DD binding to FADD. We also observed different degrees of conformational and motion changes of FADD in the Fas DD–FADD complex with different degrees of oligomerization. The increased conformational stability and the decreased degree of correlated motion of the residues in FADD in Fas DD–FADD tetramer complex were observed compared to those in Fas DD–FADD dimer complex. This study provides structural evidence for the roles of Fas DD binding to FADD and the oligomerization degree of Fas DD–FADD complex in DISC formation to signal apoptosis. Proteins 2013. © 2012 Wiley Periodicals, Inc.