Head or tail? A molecular dynamics approach to the complex structure of TNF-associated factor TRAF2

Molecular dynamics (MD)

Classical MD simulations of the TRAF2-plus trimer were performed starting with the structure obtained from AF2-multimer and using the GROMACS package [21,22] with GROMOS43A1 force field [39]. MD simulations were carried out in the NpT ensemble at 288 and 310 K by following exactly the same equilibration procedure as already reported [17,23] and here reported.

The temperature is held fixed at (288 or 310 K) using the V-rescale thermostat [24] with a coupling time of 0.1 ps. The pressure is kept constant at the reference pressure of 1 bar with a coupling time of 1 ps and an isothermal compressibility of 4.5 × 10⁻⁵ bar⁻¹, exploiting the features of the Berendsen barostat [25]. The simple point charge model is used for water molecules. The simulation box is cubic (with a side of 10.3 nm); TRAF2 monomer is immersed in 33,366 water molecules, and an appropriate number of Na⁺ or Cl⁻ counterions are added to have a wholly neutral system. MD simulations are performed at neutral pH. Periodic boundary conditions are used throughout the simulation, and the particle mesh Ewald algorithm is used to deal with the long-range Coulomb interactions [26]. A time step of 2 fs was used. A non-bond pair list cut-off of 1.0 nm was used, and the pair list was updated every ten steps. TRAF2-plus trimer is initially relaxed in vacuum via a steepest descent minimization. Then, the appropriate amount of counterions and water is added. At this point, the solvent is relaxed by a few steps (10 ps) of NVT MD at 200 K, leaving the solute untouched. Then, the whole system, solute and solvent, is equilibrated for 50 ps in the NVT ensemble at the appropriate temperature (288 or 310 K). This is the situation from which the final 500 ns long NpT MD simulation at 288 or 310 K is started.

Protein contact networks (PCNs)

The PDB files, reporting the structural information of the TRAF2-C and the TRAF2-plus proteins, have been modeled as networks, where nodes are the protein residues and the links between nodes represent the noncovalent significant intramolecular interactions.

The mathematical representation of such networks, from now on referred to PCNs, is the adjacency matrix, whose generic element is defined as:

A link exists between two nodes if their Euclidean distance lies within a given range, in our case if the distance lies in the range 4–8 Å, to account only for significant noncovalent interactions between residues.

The node degree k i is the number of links that a node has with other nodes, and in the perspective of PCNs, it describes the local stability of protein structure.

Network clustering is based on the adjacency matrix and node degree: it helps in identifying densely interconnected regions of the protein structural network, which has been demonstrated to correspond to functional domains [27,28,29].

We applied the spectral clustering method, based on the computation of the Laplacian matrix L defined as:

where D is the degree matrix (D), defined as a diagonal matrix where the nonnull elements on the diagonal d ii are the corresponding node degree k i , and A is the adjacency matrix (A). Cluster identification is based on the components of the eigenvector v 2 corresponding to the second minor eigenvalue of L [30,31].

Analysis of subunit–subunit interface

The analysis of the subunit interfaces has been performed using the COCOMAPS software [32] and the PISA service at the European Bioinformatics Institute [33]. In particular, a cutoff of 8 Å has been used to identify each couple of amino acids belonging to different interfaces, in contact with each other. The distances of such quaternary interactions have been then used to evaluate S _index as described in the text.

Analysis of the overall TRAF2-plus dynamics vs TRAF2-C

The TRAF2-plus structure at time t = 0 has been obtained using the AlphaFold2 AI system (see Methods) and superimposed to the available crystallographic file of TRAF2-C (pdb code: 1CA4), as shown in Figure 1 (right panel). TRAF2-C retains most of its overall, native, structural features in the temperature range of 15–37°C [17]. For such reason, MD simulations have been carried out both at 288 and at 310 K, and the corresponding RMSD values are reported in Figure 2.

Figure 2

RMSD time course of the TRAF2-C backbone (panel a) at 310 K (red line) and 288 K (blue line). In panel b, the RMSD of TRAF2-plus backbone at 310 K (in red) and 288 K (in blue) is shown. Insets: average RMSD values and their corresponding standard deviations. The average RMSD has been evaluated for the whole simulation (500 ns) and in the last 400, 300, 200, 100, and 50 ns, at both temperatures.

The TRAF2-C dynamics at 288 K is characterized by an almost monotonic increase between t = 0 and t = 300 ns (Figure 2), followed by oscillations around an average RMSD value of about 0.54 ± 0.02 nm (Figure 2a, inset). A more rapid increase occurs at 310 K (0 < t < 200 ns), followed by larger fluctuations, which converge to a slightly higher average RMSD value, namely, 0.68 ± 0.03 nm. The dynamics of TRAF2-plus at 310 K displays an even faster behavior, with an RMSD increase in the first ∼120 ns (Figure 2) and a final value of about ∼0.75 ± 0.03 nm (Figure 2b, inset). At 288 K, a much slower dynamics takes instead place, with a final displacement of about 0.40 ± 0.05 nm. The structural changes occurring in the secondary and tertiary structure of the two proteins at 310 K are more explicitly shown in Figure 3, where the initial (t = 0) and final (t = 500 ns) respective cartoons have been superimposed. The graphic rendering and the analysis of the secondary structure content demonstrate that the major transformations that take place in TRAF2-plus mainly concern the coiled-coil section, which is twisted with respect to the initial position, with the three long helices still fully folded. In the case of TRAF2-C, instead, a partial loss of α-helix secondary structure is observed, mainly ascribable to the partial loosening of the N-terminal tail (Figure 3).

Figure 3

Structures at 310 K of the simulated TRAF2-C (left) and TRAF2-plus (right) at t = 0 ns (blue) and t ≈ 500 ns (red). The secondary structure elements are shown in the cartoon modality (α-helices as cylinders and as flat strands) and their percentage contents quantified in the form of pie charts (helices = purple, β-structures = orange, coil = green). The little black arrow included in the picture indicates the partial unfolding of the coiled-coil tail in TRAF2-C.

The analysis of the respective monomeric structures at 310 K is reported in Figure 4 at time t = 200 ns, that is, when almost ∼95% of the RMSD change has already occurred, for both proteins (Figure 2). The structural details of the single chains have been obtained using a surface rendering, which better shows the shape of each subunit. Indeed, in this kind of representation, the N-terminal tail of the A-subunit cannot be distinguished in TRAF2-C, as it is bent toward the protein head. The N-terminal helix of subunit C is also partially unstructured, while all the monomers of TRAF2-plus maintain the fully folded tail that characterizes the protein structure at time t = 0. In order to corroborate such findings, an analysis of the principal component (PCA) of motion has been carried out at 310 K. In Figure 5, the cartoons of TRAF2-C and TRAF-plus at 0, 250, and 500 ns are reported, while the movies of both constructs along the first eigenvector are available in the files included in the supplementary materials.

Figure 4

Simulated structures of the single chains of TRAF2-plus (upper illustrations) and TRAF2-C (lower drawings) at 200 ns.

Figure 5

Simulated structures of TRAF2-plus (upper illustrations) and TRAF2-C (lower drawings) at different instants of the simulation. The three subunits, A, B, and C, are plotted in blue, red, and gray, respectively, and the secondary structure elements reported in the cartoon modality (α-helices as cylinders and β-structures as flat strands).

Such analysis put in evidence that the principal movement of TRAF2-plus involves the bending of the protein C-terminal section toward the coiled-coil helices, causing an increase in the value of the angle between the other side of the protein head and the long tail (Figure 5). In TRAF2-C, a distancing of the three short helices in the N-terminal section is evident already at 250 ns, together with the partial unfolding of the coiled-coil structure (Figure 5), diagnostic of a major structural instability of the shorter trimeric construct.

Effects on the subunit interface and on the C-terminal shape

The details on the protein quaternary structure dynamics have been obtained evaluating the average distance between each center of mass of three TRAF2 subunits (AB, BC, and CA subunits), at the two different temperatures (Figure 6). The distancing and re-approaching movements of the three subunits are particularly pronounced in TRAF2-C at 310 K, while such effects are comparatively small in the case of TRAF2-plus, suggesting that the longer coiled-coil tail reduces the protein degrees of freedom.

Figure 6

Couple distances of TRAF2-C (310 K, panel a and 288 K panel c) and TRAF2-plus (310 K panel b and 288 K, panel d) subunits as a function of time. The black, red, and blue lines correspond to the couple of AB, BC, and CA subunits, respectively.

The enhanced mobility observed in the simulation of TRAF2-C has important consequences at the level of the protein quaternary structure. In Figure 7, top views of the TRAF2-C and TRAF2-plus simulated structures are reported at t = 0, 250, and 500 ns. The graphic rendering demonstrates that in TRAF2-plus, the balance of the interactions within each couple of subunits is only marginally affected by the limited movements of the protein subunits (Figure 7IV, V and VI). The larger conformational changes that characterize the TRAF2-C dynamics display, instead, an asymmetric configuration (Figure 7II and III) and the arise of unstructured segments (Figure 7III) at the subunit interfaces with respect to TRAF2-plus (Figure 7VI). Moreover, in TRAF2-C at 250 and 500 ns, a progressive re-shape of the molecular surface, affecting the local tertiary structure at the level of the putative TNFr binding site, occurs.

Figure 7

Top view of TRAF2-C at 0, 250, and 500 ns in panels I, II, and III, respectively. The three subunits are colored in green, cyan, and purple. The cartoon rendering represents the secondary structure, while the spheres correspond to the amino acids located at the subunit interfaces. The analogous TRAF2-plus structures at 0, 250, and 500 ns are reported in panels IV, V, and VI. The black arrows in the structures at t = 0 ns indicate the putative binding site to the TNF receptor.

The extension of monomer–monomer interactions and their effects on the protein topology have been characterized by PCNs analysis, an approach that identifies the coordination of different domains and subunits in multimeric proteins [34,35,36]. In detail, the polypeptide structure is represented in terms of an amino acid network, where clusters are the sets of residues preferentially interconnected, and not necessarily coincident with the subunits of protein [37]. As shown in Figure 8, the whole head of TRAF2-plus (colored in green) acts like one, single unit, while the long tail appears to be organized in two clusters (yellow and red), distinguished by a different association degree. The segments characterized by a stronger interconnection are shown in the graphic rendering of Figure 8 and include the residues in the first portion of the coiled-coil tail, which roughly corresponds to the cluster in red reported in Figure 8a. The topology of TRAF2-C at 200 ns is completely different, as two chains form a large aggregate, while the third subunit is more independent (Figure 8c). In this case, main interactions are distributed along the interface that lies between the two clusters, involving both the helical tails and the protein heads, as shown in Figure 8d.

Figure 8

Clustering representation of TRAF2-plus (panel a) and TRAF2-C (panel c). In panels b and d, the corresponding distribution of the strongest interactions that characterize the two TRAF constructs is reported on a color scale (0–1) that ranges from blue to red.

The strained state produced at the intersection among the three monomers has also important structural consequences on the roughness of the trimer external surface. The frames obtained at intermediate states of the simulation (∼200–250 ns) suggest that changes of different extent characterize the two constructs (Figures 4 and 7). In particular, the concavity of the three bights present in the protein crystallographic structure (Figure 7, black arrows) is progressively reduced in the course of time in the case of TRAF2-C, while the minor effects occur in TRAF2-plus (Figure 7).

Evaluation of order/disorder at the level of the protein quaternary interactions

The order/disorder transition has been followed along the simulation introducing a surface index, S _index, defined as follows:

which is the sum of all the distances at time t, D _i (t), between residue pairs at the subunit interface, divided by the number of couples, n. The S _index values along the simulation are plotted in Figure 9, for both proteins.

Figure 9

Time dependence of the S _index parameter for TRAF2-C (panel a) and TRAF2-plus (panel b). The black, blue, and red symbols correspond to the AB, BC, and CA interfaces, respectively. The horizontal black line corresponds to the average value, 〈S _index〉. In the two insets, the dependence of the order parameter, P, is reported as a function of time.

The larger overall mobility of TRAF2-C (Figures 6 and 7) produces two important effects on the S _index values: (i) wider oscillations with respect to the case of TRAF2-plus and (ii) a smaller average value, <S _index>, driven by the closening of the couples BC and CA (Figure 6, panel a). Such asymmetric behavior at the subunit interfaces of TRAF2-C has been quantified introducing an order parameter, P(t), defined as the sum of the three absolute deviation values of S _index with respect to the average 〈S _index〉, at each time t:

As shown in the insets of Figure 9, P(t) is a randomly oscillating function for TRAF2-plus, while it increases up to three times its initial values in the case of TRAF2-C, as expected from a more disordered distribution of quaternary interactions at the three monomer–monomer interfaces.