Brownian dynamics simulations of glycolytic enzyme subsets with F‐actin

Abstract

Previous Brownian dynamics (BD) simulations identified specific basic residues on fructose-1,6-bisphophate aldolase (aldolase) (I. V. Ouporov et al., Biophysical Journal, 1999, Vol. 76, pp. 17-27) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (I. V. Ouporov et al., Journal of Molecular Recognition, 2001, Vol. 14, pp. 29-41) involved in binding F-actin, and suggested that the quaternary structure of the enzymes may be important. Herein, BD simulations of F-actin binding by enzyme dimers or peptides matching particular sequences of the enzyme and the intact enzyme triose phosphate isomerase (TIM) are compared. BD confirms the experimental observation that TIM has little affinity for F-actin. For aldolase, the critical residues identified by BD are found in surface grooves, formed by subunits A/D and B/C, where they face like residues of the neighboring subunit enhancing their electrostatic potentials. BD simulations between F-actin and aldolase A/D dimers give results similar to the native tetramer. Aldolase A/B dimers form complexes involving residues that are buried in the native structure and are energetically weaker; these results support the importance of quaternary structure for aldolase. GAPDH, however, placed the critical residues on the corners of the tetramer so there is no enhancement of the electrostatic potential between the subunits. Simulations using GAPDH dimers composed of either S/H or G/H subunits show reduced binding energetics compared to the tetramer, but for both dimers, the sets of residues involved in binding are similar to those found for the native tetramer. BD simulations using either aldolase or GAPDH peptides that bind F-actin experimentally show complex formation. The GAPDH peptide bound to the same F-actin domain as did the intact tetramer; however, unlike the tetramer, the aldolase peptide lacked specificity for binding a single F-actin domain.

Brownian dynamics of interactions between glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mutants and F‐actin

“…However, the error bars on the corresponding free energy profiles (graph not shown) overlap each other, indicating that the difference is not statistically significant. This observation was also seen in the work of Lowe et al 35 in which the binding of GAPDH dimers and F-actin was studied. The dimer results indicated that both the G/H dimer and the S/H dimer showed similar binding affinities towards F-actin and had similar free energy profiles.…”

“…This new region of positive potential only becomes apparent when the four critical lysines are mutated, and because it is less exposed, it cannot interact as strongly as the original surface patch. Although the Clarke and Sheedy peptide can compete with native enzyme 34 and BD has been able to reproduce the competitive nature of the peptide binding, 35 when left within the intact protein, the region of this peptide cannot compete because it is much less exposed due to the quaternary structure of the enzyme. It is clear that the surface patch formed by the four BD identified lysines is an important electrostatic feature needed for GADPH to bind Factin.…”

“…This has also been observed by the work of Lowe et al involving the study of interactions of aldolase and aldolase mutants with F-actin 34 and also of aldolase and GAPDH dimers and peptides with F-actin. 35 …”

Functional Diversity

“…substitutions with alanyl residues) did not have a large effect on the average electrostatic binding energy, suggesting that the lysine residues act in concert. Additionally, the binding unit of GAPDH appears to be the GAPDH dimer [22]. The proposed mode of interaction is that two subunits of GAPDH interact with two adjacent subunits of F-actin, although a one to one interaction is still plausible with the remaining quaternary structure not forming contacts.…”

“…It is interesting that the actin filament (Fig. 4.2) has a diameter of about 80 Å and a repeat distance along the axis of about 55 Å .The binding mode of GAPDH to actin involves two GAPDH subunits that bind to two adjacent actin subunits [22]. The binding of one pair of subunits on GAPDH allows the adjacent pair to interact with another actin filament resulting in crosslinking of the filaments.…”