Divalent Cations Can Induce the Exposure of GroEL Hydrophobic Surfaces and Strengthen GroEL Hydrophobic Binding Interactions: NOVEL EFFECTS OF Zn2+ GroEL INTERACTIONS

Bill T Brazil,Jesse Ybarra,Paul M Horowitz

doi:10.1074/jbc.273.6.3257

Bill T Brazil, Jesse Ybarra + Show 1 more

Open Access

https://doi.org/10.1074/jbc.273.6.3257

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Abstract

Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca2+, Mg2+, Mn2+, and Zn2+) significantly increase hydrophobic exposure on GroEL, whereas monovalent cations (K+ and Na+) have little effect. Zn2+ always induced the largest amount of hydrophobic exposure on GroEL. By using a new method based on interactions of GroEL with octyl-Sepharose, it was demonstrated that Zn2+ binding strengthens GroEL hydrophobic binding interactions and increases the efficiency of substrate release upon the addition of MgATP and GroES. The binding of 4, 4'-bis(1-anilino-8-naphthalenesulfonic acid) to GroEL in the presence of Zn2+ has a Kd congruent with 1 microM, which is similar to that observed previously for the GroEL 4, 4'-bis(1-anilino-8-naphthalenesulfonic acid) complex. Urea denaturation, sedimentation velocity ultracentrifugation, and electron microscopy revealed that the quaternary structure of GroEL in the presence of Zn2+ had a stability and morphology equivalent to unliganded GroEL. In contrast, circular dichroism suggested some loss in both alpha-helical and beta-sheet secondary structure in the presence of Zn2+. These data suggest that divalent cations can modulate the amount of hydrophobic surface presented by GroEL. Furthermore, the influence of Zn2+ on GroEL hydrophobic surface exposure as well as substrate binding and release appears to be distinct from the stabilizing effects of Mg2+ on GroEL quaternary structure.

Highlights

Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca2؉, Mg2؉, Mn2؉, and Zn2؉) significantly increase hydrophobic exposure on GroEL, whereas monovalent cations (K؉ and Na؉) have little effect
We demonstrate the importance of divalent cations in altering the apparent amount of hydrophobic surface presented by GroEL
GroEL binds a variety of polypeptides in their non-native states (3, 20 –21)

Summary

Introduction

Fluorescent and non-fluorescent probes have been used to show that divalent cations (Ca2؉, Mg2؉, Mn2؉, and Zn2؉) significantly increase hydrophobic exposure on GroEL, whereas monovalent cations (K؉ and Na؉) have little effect. Circular dichroism suggested some loss in both ␣-helical and ␤-sheet secondary structure in the presence of Zn2؉ These data suggest that divalent cations can modulate the amount of hydrophobic surface presented by GroEL. The influence of Zn2؉ on GroEL hydrophobic surface exposure as well as substrate binding and release appears to be distinct from the stabilizing effects of Mg2؉ on GroEL quaternary structure. It has been suggested that GroEL binds the compact folding intermediates (molten globules) of proteins produced in the course of folding [7], and recent calorimetric studies have demonstrated that hydrophobic interactions are an important driving force for the association of substrate proteins with GroEL [8]. This implies that most of the hydrophobic residues used for polypeptide binding are either buried or that the negatively charged bis-ANS molecule does not have access to the hydrophobic residues, due to the high net negative charge on GroEL (Ϫ266 per 14-mer)

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