RESUMO
Centralities determined from Residue Interaction Networks (RIN) in proteins have been used to predict aspects of their structure and dynamics. Here, we correlate the Eigenvector Centrality (Ec) with the rate constant for thermal denaturation (kden) of the HisF protein from Thermotoga maritima based on 12 single alanine substitution mutants. The molecular basis for this correlation was further explored by studying a mutant containing a replacement of a high Ec residue, Y182A, which displayed increased kden at 80 °C. The crystallographic structure of this mutant showed few changes, mostly in two flexible loops. The 1H-15N -HSQC showed only subtle changes of cross peak positions for residues located near the mutation site and scattered throughout the structure. However, the comparison of the RIN showed that Y182 is the vertex of a set of high centrality residues that spreads throughout the HisF structure, which is lacking in the mutant. Cross-correlation displacements of Cα calculated from a molecular dynamics simulation at different temperatures showed that the Y182A mutation reduced the correlated movements in the HisF structure above 70 °C. 1H-15N NMR chemical shift covariance using temperature as perturbation were consistent with these results. In conclusion the increase in temperature drives the structure of the mutant HisF-Y182A into a less connected state, richer in non-concerted motions, located predominantly in the C-terminal half of the protein where Y182 is placed. Conversely, wild-type HisF responds to increased temperature as a single unit. Hence the replacement of a high Ec residue alters the distribution of thermal energy through HisF structure.
Assuntos
Proteínas , Thermotoga maritima , Modelos Moleculares , Conformação Proteica , Thermotoga maritima/genéticaRESUMO
BACKGROUND: Understanding the determinants of protein thermostability is very important both from the theoretical and applied perspective. One emerging view in thermostable enzymes seems to indicate that a salt bridge/charged residue network plays a fundamental role in their thermostability. METHODS: The structure of alkaline phosphatase (AP) from Thermus thermophilus HB8 was solved by X-ray crystallography at 2.1 Å resolution. The obtained structure was further analyzed by molecular dynamics studies at different temperatures (303 K, 333 K and 363 K) and compared to homologous proteins from the cold-adapted organisms Shewanella sp. and Vibrio strain G15-21. To analyze differences in measures of dynamic variation, several data reduction techniques like principal component analysis (PCA), residue interaction network (RIN) analysis and rotamer analysis were used. Using hierarchical clustering, the obtained results were combined to determine residues showing high degree dynamical variations due to temperature jumps. Furthermore, dynamic cross correlation (DCC) analysis was carried out to characterize networks of charged residues. RESULTS: Top clustered residues showed a higher propensity for thermostabilizing mutations, indicating evolutionary pressure acting on thermophilic organisms. The description of rotamer distributions by Gini coefficients and Kullback-Leibler (KL) divergence both revealed significant correlations with temperature. DCC analysis revealed a significant trend to de-correlation of the movement of charged residues at higher temperatures. SIGNIFICANCE: The de-correlation of charged residues detected in Thermus thermophilus AP, highlights the importance of dynamic electrostatic network interactions for the thermostability of this enzyme.
Assuntos
Fosfatase Alcalina/química , Temperatura Alta , Thermus thermophilus/enzimologia , Sequência de Aminoácidos , Cristalografia por Raios X , Estabilidade Enzimática , Ligação de Hidrogênio , Simulação de Dinâmica Molecular , Conformação Proteica , Homologia de SequênciaRESUMO
Network structural analysis, known as residue interaction networks or graphs (RIN or RIG, respectively) or protein structural networks or graphs (PSN or PSG, respectively), comprises a useful tool for detecting important residues for protein function, stability, folding and allostery. In RIN, the tertiary structure is represented by a network in which residues (nodes) are connected by interactions (edges). Such structural networks have consistently presented a few central residues that are important for shortening the pathways linking any two residues in a protein structure. To experimentally demonstrate that central residues effectively participate in protein properties, mutations were directed to seven central residues of the ß-glucosidase Sfßgly (ß-D-glucoside glucohydrolase; EC 3.2.1.21). These mutations reduced the thermal stability of the enzyme, as evaluated by changes in transition temperature (Tm ) and the denaturation rate at 45 °C. Moreover, mutations directed to the vicinity of a central residue also caused significant decreases in the Tm of Sfßgly and clearly increased the unfolding rate constant at 45 °C. However, mutations at noncentral residues or at surrounding residues did not affect the thermal stability of Sfßgly. Therefore, the data reported in the present study suggest that the perturbation of the central residues reduced the stability of the native structure of Sfßgly. These results are in agreement with previous findings showing that networks are robust, whereas attacks on central nodes cause network failure. Finally, the present study demonstrates that central residues underlie the functional properties of proteins.