Correlation between conformational stability of the ternary enzyme–substrate complex and domain closure of 3‐phosphoglycerate kinase

Abstract

The experimental and theoretical studies that led to our present understanding of protein structural changes and their role in enzyme function were mostly carried out on small single-domain proteins [1]. Most enzymes, however, are built of several domains. The mechanism of transmission of molecular interactions over large distances (e.g. from one domain to the other) within the molecule, the role of substrates in 3-Phosphoglycerate kinase (PGK) is a typical two-domain hinge-bending enzyme with a well-structured interdomain region. The mechanism of domain-domain interaction and its regulation by substrate binding is not yet fully understood. Here the existence of strong cooperativity between the two domains was demonstrated by following heat transitions of pig muscle and yeast PGKs using differential scanning microcalorimetry and fluorimetry. Two mutants of yeast PGK containing a single tryptophan fluorophore either in the N-or in the C-terminal domain were also studied. The coincidence of the calorimetric and fluorimetric heat transitions in all cases indicated simultaneous, highly cooperative unfolding of the two domains. This cooperativity is preserved in the presence of substrates: 3-phosphoglycerate bound to the N domain or the nucleotide (MgADP, MgATP) bound to the C domain increased the structural stability of the whole molecule. A structural explanation of domain-domain interaction is suggested by analysis of the atomic contacts in 12 different PGK crystal structures. Well-defined backbone and side-chain H bonds, and hydrophobic and electrostatic interactions between side chains of conserved residues are proposed to be responsible for domain-domain communication. Upon binding of each substrate newly formed molecular contacts are identified that firstly explain the order of the increased heat stability in the various binary complexes, and secondly describe the possible route of transmission of the substrate-induced conformational effects from one domain to the other. The largest stability is characteristic of the native ternary complex and is abolished in the case of a chemically modified inactive form of PGK, the domain closure of which was previously shown to be prevented

A Spring-loaded Release Mechanism Regulates Domain Movement and Catalysis in Phosphoglycerate Kinase

“…Therefore, the closed conformation is "primed" on substrate binding. Substrate binding may also assist in the interdomain communication and domain closure by the aid of well described interaction networks (17)(18)(19) and acts in concert with the internal dynamics of the protein. Therefore, as soon as the domains come together, the salt bridges are formed, and the closed conformation will be stabilized sufficiently for catalysis to occur.…”

“…In the closed conformation of PGK, the two domains reorient by 33°, compared with the open form, about central hinge regions to bring the substrates together. Much effort has been put into elucidating the mechanism of this hinge bending and the role that substrate binding plays in inducing the conformational changes needed for catalysis to occur (15)(16)(17)(18)(19).…”

Protein Stability, Folding and Misfolding in Human PGK1 Deficiency

“…The domains are separated by a deep cleft and linked by two alpha-helices (α-helix 7 and α-helix 14) [59,60]. Contacts between the two domains are formed through hydrophobic interactions and hydrogen bonds [61,62]. The enzyme can adopt different conformational states and transition between these states can be triggered by ligand binding.…”

“…Thus, it was suggested that upon binding of the two substrates, a hinge-bending motion converts the enzyme to the closed form to allow the substrates to come into contact. The domain closure requires the concerted action of both substrates and takes place under a strong cooperativity between the two domains [60,62]. …”

“…In an evolutionary context, we must indicate that PGK enzymes from other mesophiles (such as yeast and pig) show similarly high values of T m and E a [62]. An interesting possibility is that evolution may have selected some energetic features ( i.e.…”