Molten Globule-like State of Peanut Lectin Monomer Retains Its Carbohydrate Specificity

Abstract

A central question in biological chemistry is the minimal structural requirement of a protein that would determine its specificity and activity, the underlying basis being the importance of the entire structural element of a protein with regards to its activity vis à vis the overall integrity and stability of the protein. Although there are many reports on the characterization of protein folding/ unfolding intermediates, with considerable secondary structural elements but substantial loss of tertiary structure, none of them have been reported to show any activity toward their respective ligands. This may be a result of the conditions under which such intermediates have been isolated or due to the importance of specific structural elements for the activity. In this paper we report such an intermediate in the unfolding of peanut agglutinin that seems to retain, to a considerable degree, its carbohydrate binding specificity and activity. This result has significant implications on the molten globule state during the folding pathway(s) of proteins in general and the quaternary association in legume lectins in particular, where precise subunit topology is required for their biologic activities.Despite their preponderance in biologic systems, studies on the folding pathways of oligomeric proteins are less common and information regarding the same, meager. The folding process for oligomeric proteins is more complex as the acquisition of the quaternary structure entails both, the intramolecular refolding of the individual polypeptide chains and the simultaneous intermolecular interactions between the various subunits. Hence, elucidation of the hierarchy of events occurring during the denaturation of an oligomeric protein provides important means to delineate such a process.Legume lectins, a class of highly homologous, carbohydratebinding proteins exhibit the same jelly roll tertiary structural fold but differ considerably in their ligand specificity and quaternary structures (1, 3). They can hence be considered to be "natural mutants" of quaternary structures. However, apart from their agglutinating activity, the role of oligomerization in lectins is not clearly understood (1-11). Peanut (Arachis hypogea) agglutinin (PNA), 1 a homotetrameric nonglycosylated protein, violates an important principle of quaternary association in globular proteins, a unique case of a tetramer without a 4-fold or 222 symmetry (10, 11). Unlike most other legume lectins, which dissociate and unfold as single entities (12-14), we describe here its chaotrope (guanidine hydrochloride, GdnHCl) induced unfolding that consists of a substantially unfolded monomeric species, as an intermediate, which retains its carbohydrate specificity despite a considerable loss of tertiary structure. Existence of such a molten globule-like structure with retention of binding activity is perhaps unprecedented so far. Moreover, the occurrence of such a species for PNA suggests that the monomers of legume lectins are competent to bind sugars and oligomerization appear...

Thermodynamic analysis of three state denaturation of Peanut Agglutinin

“…Hence, it has been described as a molten globule like state. This molten globule like state is monomeric and retains its carbohydrate binding specificity (15). The denaturation profile is slightly different from our earlier study (24).…”

“…PNA is a homotetrameric non-glycosylated legume lectin with a very unusual quaternary structure described as 'open' quaternary structure (14,15). The tertiary structural motif of a PNA monomer can be described as 'jelly roll' fold (16,17), which consists of a 6-stranded flat back b sheet and a 7-stranded curved front b sheet.…”

Disulfide bond formation through Cys186 facilitates functionally relevant dimerization of trimeric hyaluronan‐binding protein 1 (HABP1)/p32/gC1qR

“…The state of HABP1 with the larger hydrodynamic radius may be different from the crystal structure, as the changes associated with the addition of trace amounts of bivalent cations may represent a structural state in which this cysteine is exposed to the solvent, facilitating inter-trimeric disul®de bond formation. HABP1 with a larger hydrodynamic radius and solvent-exposed cysteine residue under physiological conditions may correspond to the expanded structure [33].…”

Kinetics and the Mechanism of Interaction of the Endoplasmic Reticulum Chaperone, Calreticulin, with Monoglucosylated (Glc1Man9GlcNAc2) Substrate

“…In the latter, multivalency increases not only their binding affinities but also their specificities and physiological responses such as cell agglutination, mitogenicity, etc. (51)(52)(53)(54). A wealth of information on the affinities of plant lectin-saccharide/glycopeptide interaction is available, which in turn has been exploited for the isolation and the structural characterization of the latter by affinity chromatography (55)(56)(57).…”