Mechanisms of Intramolecular Communication in a Hyperthermophilic Acylaminoacyl Peptidase: A Molecular Dynamics Investigation

Abstract

Protein dynamics and the underlying networks of intramolecular interactions and communicating residues within the three-dimensional (3D) structure are known to influence protein function and stability, as well as to modulate conformational changes and allostery. Acylaminoacyl peptidase (AAP) subfamily of enzymes belongs to a unique class of serine proteases, the prolyl oligopeptidase (POP) family, which has not been thoroughly investigated yet. POPs have a characteristic multidomain three-dimensional architecture with the active site at the interface of the C-terminal catalytic domain and a β-propeller domain, whose N-terminal region acts as a bridge to the hydrolase domain. In the present contribution, protein dynamics signatures of a hyperthermophilic acylaminoacyl peptidase (AAP) of the prolyl oligopeptidase (POP) family, as well as of a deletion variant and alanine mutants (I12A, V13A, V16A, L19A, I20A) are reported. In particular, we aimed at identifying crucial residues for long range communications to the catalytic site or promoting the conformational changes to switch from closed to open ApAAP conformations. Our investigation shows that the N-terminal α1-helix mediates structural intramolecular communication to the catalytic site, concurring to the maintenance of a proper functional architecture of the catalytic triad. Main determinants of the effects induced by α1-helix are a subset of hydrophobic residues (V16, L19 and I20). Moreover, a subset of residues characterized by relevant interaction networks or coupled motions have been identified, which are likely to modulate the conformational properties at the interdomain interface.

Structural investigation of the cold-adapted acylaminoacyl peptidase from Sporosarcina psychrophila by atomistic simulations and biophysical methods

“…Additionally, in the monomeric structure, the N-terminal helix α1 does not feature any highly populated paths of interactions to the rest of the protein. This is a marked difference with the hyperthermophilic ApAAP variant, in which the hydrophobic residues of helix α1 mediate long-range interactions to the N-terminal domain and the catalytic site [36]. Moreover, a larger number of salt-bridge networks characterize the ApAAP interface between the two domains [36].…”

“…This is a marked difference with the hyperthermophilic ApAAP variant, in which the hydrophobic residues of helix α1 mediate long-range interactions to the N-terminal domain and the catalytic site [36]. Moreover, a larger number of salt-bridge networks characterize the ApAAP interface between the two domains [36]. The main hubs are characterized by a ΔΔG value greater than 0.5 kcal/mol upon alanine mutations (Fig.…”

“…Indeed, in the case of a pair of residues with multiple atoms involved in the interaction only one edge was considered. Interactions with persistence lower than 20% were discarded because they are poorly representative of the structural properties of the protein, as previously observed in other cases that have been studied [31,36,90,91].…”

“…In particular, dynamically coupled residues are involved in protein structural stability and activity [26][27][28][29][30][31][32] as also shown by mutational studies [30,31,[33][34][35][36]. NMR data indicate that the motions on the nanosecond (ns) or longer timescales are equally important.…”

Alteration of substrate specificities of thermophilic α/β hydrolases through domain swapping and domain interface optimization

“…Using its CSU (contacts of structural units) server, we analyzed the interactions of the residues on the domain boundaries of apAPH and AFEST involved in, and then chose the residues with the least contacts as the recombination sites. Some researchers have shown that the interdomain interactions are crucial to the conformation and consequently stability and function of enzymes [8,28]. The propeller domain of apAPH and cap domain of AFEST are very different in sequence, size, and structure.…”