Structure of GroEL in Complex with an Early Folding Intermediate of Alanine Glyoxylate Aminotransferase

Armando Albert,Cristina Yunta,Rocío Arranz,Álvaro Peña,Eduardo Salido,José María Valpuesta,Jaime Martín-Benito

doi:10.1074/jbc.m109.062471

Abstract

Primary hyperoxaluria type 1 is a rare autosomal recessive disease caused by mutations in the alanine glyoxylate aminotransferase gene (AGXT). We have previously shown that P11L and I340M polymorphisms together with I244T mutation (AGXT-LTM) represent a conformational disease that could be amenable to pharmacological intervention. Thus, the study of the folding mechanism of AGXT is crucial to understand the molecular basis of the disease. Here, we provide biochemical and structural data showing that AGXT-LTM is able to form non-native folding intermediates. The three-dimensional structure of a complex between the bacterial chaperonin GroEL and a folding intermediate of AGXT-LTM mutant has been solved by cryoelectron microscopy. The electron density map shows the protein substrate in a non-native extended conformation that crosses the GroEL central cavity. Addition of ATP to the complex induces conformational changes on the chaperonin and the internalization of the protein substrate into the folding cavity. The structure provides a three-dimensional picture of an in vivo early ATP-dependent step of the folding reaction cycle of the chaperonin and supports a GroEL functional model in which the chaperonin promotes folding of the AGXT-LTM mutant protein through forced unfolding mechanism.

Highlights

Hereditary disease primary hyperoxaluria type 1 (PH1, Online Mendelian Inheritance in Man (OMIM) 259900)
Coexpression of GroEL-GroES and AGXT-LTM Increases the Solubility of the Mutant Protein and Results in the Formation of a GroEL1⁄7AGXT-LTM Stable Complex—We first measured the AGXT activity from the soluble extracts when AGXT-LTM was expressed in E. coli either in the absence or presence of
Our data show that the AGXT activity of E. coli lysates increases when mutant AGXT-LTM is expressed in presence of the GroEL-GroES system (Fig. 1A)

Summary

Introduction

Hereditary disease primary hyperoxaluria type 1 (PH1, Online Mendelian Inheritance in Man (OMIM) 259900). Working with a total of 11,011 and 8,303 images (for nucleotide-free and ATP-bound samples, respectively) these initial rough models were refined imposing 7-fold or no symmetry using EMAN [23] and SPIDER software subsequently [24]. This result indicates that GroEL-GroES coexpression improves, in vivo, the yield of soluble and active AGXT-LTM mutant protein.

Results

Conclusion