A study of collective atomic fluctuations and cooperativity in the U1A-RNA complex based on molecular dynamics simulations

Abstract

Cooperative interactions play an important role in recognition and binding in macromolecular systems. In this study, we find that cross-correlated atomic fluctuations can be used to identify cooperative networks in a protein-RNA system. The dynamics of the RRM-containing protein U1A-stem loop 2 RNA complex have been calculated theoretically from a 10 ns molecular dynamics (MD) simulation. The simulation was analyzed by calculating the covariance matrix of all atomic fluctuations. These matrix elements are then presented in the form of a two-dimensional grid, which displays fluctuations on a per residue basis. The results indicate the presence of strong, selective cross-correlated fluctuations throughout the RRM in U1A-RNA. The atomic fluctuations correspond well with previous biophysical studies in which a multiplicity of cooperative networks have been reported and indicate that the various networks identified in separate individual experiments are fluctuationally correlated into a hyper-network encompassing most of the RRM. The calculated results also correspond well with independent results from a statistical covariance analysis of 330 aligned RRM sequences. This method has significant implications as a predictive tool regarding cooperativity in the protein-nucleic acid recognition process.

Figure 1

Amino acid sequence of the N-terminal RRM of the protein U1A. Residue numbers are labeled and the secondary structure is indicated above the sequence.

Figure 2

Tertiary structure of the N-terminal RRM of U1A bound to stem-loop 2 RNA (Oubridge et al., 1994). Inset: Nucleic acid sequence of stem-loop 2 RNA with nucleotides recognized by U1A for binding highlighted in red.

Figure 3

Root-mean-square deviation (RMSD) of the protein backbone and all RNA atoms from the equilibrated structure calculated over the 10 ns simulation of the U1A-SL2 RNA complex.

Figure 4

Root-mean-square fluctuations (RMSF) computed on a residue or nucleotide basis with reference to the average structure calculated over the 10 ns simulation of the U1A-SL2 RNA complex.

Figure 5

Cooperative networks of interactions between residues contributing to binding and specificity in U1A-RNA. (A) Network 1: Interactions from Tyr13 through C5 and Phe56 through A6 and C7, RNA to the C-terminal residues Lys96 – Phe101. (B) Network 2: Interactions from Tyr13 to Gln54 and the loop 3 residues through the RNA closing C·G base pair and A1. (C) Network 3: Interactions from Gly53 through loop 3 through loop 1, loops 1 and 3 through RNA to the TDS linker.

Figure 6

Calculated dynamical cross-correlation map (DCCM) for U1A-RNA. Magnitudes of calculated cross-correlations are indicated via a grey scale with black, slate gray and light gray regions corresponding to strong (C_ij = ± 0.75–1.00), moderate (C_ij = ± 0.50–0.75), and weak (C_ij = ± 0.25–0.50) correlated fluctuations, respectively. Red and blue squares have been drawn to illustrate positive and negative protein-protein secondary structure intersections, respectively. Orange (positive correlations) and green (negative correlations) lines have been drawn to distinguish between RNA stem and loop nucleotides. Additional details can be found in the text.

Figure 7

Calculated DCCMs for U1A-RNA indicating residues implicated in cooperativity studies. (A) Red, orange and yellow boxes drawn indicate correlated fluctuations of the residues implicated in networks 1, 2 and 3, respectively. (B) Boxes drawn indicate correlated fluctuations of all residues implicated in the three cooperative networks with each other.

Figure 8

Calculated DCCM for U1A-RNA. Yellow boxes indicate residues revealed to exhibit high covariance from Crowder et al. (2001) statistical covariance analysis of 330 RRM-containing proteins.

Figure 9

Cooperative interactions related to the U1A-RNA binding interface predicted from both MD calculated fluctuations and statistical covariance analysis for U1A. Residues directly in contact with RNA are indicated by an asterisk. The magnitude of fluctuational covariance between residues corresponds to the line coloring: purple: C_ij = ± 0.75–1.00, blue: C_ij = ± 0.50–0.75, green: C_ij = ± 0.25–0.50.