Discrimination of ATP, ADP, and AMPPNP by Chaperonin GroEL

Abstract

The double ring chaperonin GroEL binds unfolded protein, ATP, and GroES to the same ring, generating the cis ternary complex in which folding occurs within the cavity capped by GroES (cis folding). The functional role of ATP, however, remains unclear since several reports have indicated that ADP and AMPPNP (5 -adenylyl-␤,␥-imidodiphosphate) are also able to support the formation of the cis ternary complex and the cis folding. To minimize the effect of contaminated ATP and adenylate kinase, we have included hexokinase plus glucose in the reaction mixtures and obtained new results. In ADP and AMPPNP, GroES bound quickly to GroEL but bound very slowly to the GroEL loaded with unfolded rhodanese or malate dehydrogenase. ADP was unable to support the formation of cis ternary complex and cis folding. AMPPNP supported cis folding of malate dehydrogenase to some extent but not cis folding of rhodanese. In the absence of hexokinase, apparent cis folding of rhodanese and malate dehydrogenase was observed in ADP and AMPPNP. Thus, the exclusive role of ATP in generation of the cis ternary complex is now evident.The bacterial chaperonin system consisting of GroEL and GroES facilitates folding of other proteins using the energy of ATP hydrolysis. GroEL is composed of 14 identical 57-kDa subunits, each containing a site for binding and hydrolysis of ATP. Seven GroEL subunits are arranged in a heptamer ring forming a central cavity, and two heptamer rings are stacked back to back. GroES is a dome-shaped, single heptamer ring of 10-kDa subunits. GroEL binds a wide range of unfolded proteins at the apical cavity surface and subsequently binds ATP and GroES to the same GroEL ring (the cis ring, a GroEL heptamer ring that binds to GroES), producing the complex consisting of GroEL, unfolded protein, and GroES (the cis ternary complex). Since the residues of the GroEL apical surface involved in GroES binding are mostly overlapped with substrate protein binding (1), GroES binding results in encapsulating unfolded protein into the enlarged cavity of the cis GroEL ring capped by GroES (the cis cavity) (2). The unfolded protein initiates folding in the cis cavity without a risk of aggregation (the cis folding). ATP hydrolysis in the cis ring and subsequent ATP binding to the opposite side of GroEL ring (the trans ring) induce the release of GroES, ADP, and substrate protein (whether folded or not) from the cis ring (3, 4). When unfolded protein is added to the GroEL-GroES complex, it binds to the trans ring, and its folding is arrested (2). Binding and release from the trans ring enable the folding for some proteins (2), especially large ones that are too large to be encapsulated in the cis cavity, by lowering the concentration of aggregation-prone folding intermediates in bulk solution (5). In contrast, the stringent substrate proteins for chaperonin fold efficiently by the cis folding in the presence of ATP (3, 6).However, it should be noted that the functional significance of ATP for the cis ternary complex formation is unclear yet. I...

Substrate polypeptide presents a load on the apical domains of the chaperonin GroEL

“…Solutions contained 0.02 M chaperonin or 0.02 M chaperonin-polypeptide binary complexes in buffer A; 0.1 M GroES and 1 mM nucleotide were added and mixed manually. When ADP was used, 0.01 units͞ l hexokinase and 100 mM glucose were added to hydrolyze any contaminating ATP (37). Where indicated, 0.2 mM KAl(SO 4 ) 2 and 5 mM KF were also added.…”

BeF Stops the Chaperonin Cycle of GroEL-GroES and Generates a Complex with Double Folding Chambers

“…GroES labeled with Cy3 (GroES Cy3 ) was prepared as described (14) in a stoichiometry of ϳ0.7 Cy3 dye molecules/GroES 7-mer. GroEL saturated with denatured rhodanese was prepared as described (21). Briefly, 4 M rhodanese was heatdenatured at 60°C for 15 min in HKM buffer (20 mM HEPES-KOH, pH 7.4, 100 mM KCl, 5 mM MgCl 2 ) containing 1 mM dithiothreitol (DTT) and 1 M GroEL.…”

The Intermediate Domain Defines Broad Nucleotide Selectivity for Protein Folding in Chlamydophila GroEL1

“…In addition, EC GroEL even enhances protein refolding of some substrates in the presence of the ADP and ATP analogs ATP␥S and AMP-PNP (13-15). However, it was shown that stringent substrates are not refolded by EC GroEL in the presence of the unhydrolyzed species ADP, ATP␥S, and AMP-PNP (16). In the present study, METF, the obligate substrate for EC GroEL (2,25,26), was recovered both by EC GroEL/EC GroES and by CP GroEL1/CP GroES in the presence of ATP, CTP, and UTP (Fig.…”

“…METF refolding assay was carried out as described in our previous study (19) with a modification. When testing ADP in the refolding reaction, ATP contamination was eliminated as much as possible by hexokinase/glucose treatment (16).…”

“…In characterizing CP GroEL1, we took into consideration the fact that EC GroEL assists refolding of these enzymes in the presence of ATP (2, 10 -12). Furthermore, because EC GroEL has been found to enhance refolding of some non-obligate substrates even in the presence of ADP and ATP analogs ATP␥S and AMP-PNP (13)(14)(15)(16) and CTP and UTP (17), the protein folding assay was also conducted in the presence of various nucleotides (i.e. CTP, UTP, GTP, and their di-or monophosphates) to elucidate the nucleotide selectivity of CP GroEL1-as well as EC GroEL-assisted protein refolding.…”