The Crystal Structure of Protein MJ1225 from Methanocaldococcus jannaschii Shows Strong Conservation of Key Structural Features Seen in the Eukaryal γ-AMPK

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Abstract

In mammals, 5′-AMP-activated protein kinase (AMPK) is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (α) and two regulatory subunits (β and γ). The γ-subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex. We describe herein the crystal structure of protein MJ1225 from Methanocaldococcus jannaschii complexed to AMP, ADP, and ATP. Our data provide evidence of a strong conservation of the key functional features seen in the γ-subunit of the eukaryotic AMPK, and more importantly, it reveals a novel AMP binding site, herein denoted as site E, which had not been previously described in cystathionine β-synthase domains so far. Site E is located in a small cavity existing between the α-helices structurally equivalent to those disrupting the internal symmetry of each Bateman domain in γ-AMPKs and shows striking similarities with a symmetry-related crevice of the mammalian enzyme that hosts the pathological mutation N488I.

Introduction

The cystathionine β-synthase (CBS) domains are 60-residue-long motifs (IPR000644, InterPro database) that usually occur as tandem pairs (two or four copies) in stand-alone proteins or are fused to different protein domains. Their importance is underlined by the fact that mutations within their sequence cause several hereditary diseases in humans. Thus, they can be considered as promising targets for the development of novel drugs.1 Comparison among CBS domains of isofunctional proteins from different species shows a highly conserved fold despite the low degree of sequence similarity.2, 3 Recently, the crystal structures of the complexes of several CBS-domain-containing proteins with nucleotides, such as CLC5-ADP/ATP4 or AMPK-ATP/AMP/ADP/ZMP,5, 6, 7, 8, 9 have been reported. These studies have provided crucial information that aids in understanding the architecture in binding to nucleotides. CBS domains are unusually abundant in archaea, although scarce information about their function has been reported. Therefore, organisms such as the hyperthermophile Methanocaldococcus jannaschii10 offer excellent models for the characterization of the adenosyl binding site of CBS domains. The genome of M. jannaschii encodes 15 CBS domain proteins‡, which differ significantly in their composition and presumably in their abilities to bind to different ligands. A close examination of their amino acid sequences reveals the presence of two different groups: (i) one with very short sequences and not fused to other domains (as in the MJ0729 protein)11, 12 and (ii) another with longer amino acid sequences that are fused to other protein motifs.13 Only two of these latter proteins (so-called MJ1404 and MJ1225) contain four CBS domains in tandem, as in the γ-subunit of γ-AMP-activated protein kinase (AMPK) and its homologs.5

AMPK plays a key role in regulation of metabolism and acts as a central regulator of energy homeostasis of eukaryotic cells, such that an increase in the cellular AMP/ATP ratio results in activation of AMPK, which then acts to inhibit anabolic pathways and activate catabolic pathways.14, 15, 16, 17 AMPK also has important roles in the regulation of cell growth and proliferation and in the establishment and maintenance of cell polarity15 and has recently been postulated as a glycogen sensor.18, 19 These important functions have rendered AMPK an important drug target for obesity, type 2 diabetes, and cancer treatments.20, 21 In mammals, AMPK is a heterotrimeric protein composed of a catalytic serine/threonine kinase subunit (α) and two regulatory subunits (β and γ), with each subunit belonging to a larger isoform family comprising α1, α2, β1, β2, γ1, γ2, and γ3, exhibiting varying tissue and subcellular expression.17 The γ-AMPK subunit senses the intracellular energy status by competitively binding AMP and ATP and is thought to be responsible for allosteric regulation of the whole complex.22 Recent findings suggest that ADP might also play a role in AMPK regulation.7 The importance of the γ-subunit is underlined by the range of hereditary diseases in humans associated with mutations in its sequence, including hypertrophic cardiomiopathy, Wolff–Parkinson–White syndrome and glycogen storage in the skeletal muscle.23, 24, 25, 26, 27, 28, 29, 30 Structurally, the three isoforms of γ-AMPK differ in their N-terminal sequences but share the presence of four conserved CBS domains (two Bateman domains) that are connected by short peptide chains ranging between 20 and 25 amino acid residues (Fig. 1).5, 6 However, although several proteins fulfill similar structural features suggesting that a prokaryotic homolog of γ-AMPK might exist (Fig. 1), no prokaryotic AMPK has been described or structurally characterized so far. Some authors have recently pointed out that it is possible that nonhomologous (or undetectably homologous) kinase domains interact with and are regulated by stand-alone CBS domain proteins in prokaryotes, forming energy-sensing kinase complexes that serve physiological roles analogous to those of AMPK or, alternatively, that stand-alone CBS domain proteins may interact directly with metabolic enzymes, without making use of a central intermediary kinase.31 On the basis of a detailed sequence analysis of those residues constituting the ligand binding sites in known γ-AMPKs in comparison with those present in CBS-domain-containing proteins from M. jannaschii, as well as from the observation of basic structural features, we recently proposed protein MJ1225 as a putative archaeal homolog of γ-AMPK.32 Accordingly, MJ1225 was overexpressed, purified, and crystallized as a first step towards its three-dimensional and functional characterization.32 Here, we report crystal structures of the native protein MJ1225 complexed to AMP, ADP, and ATP (or to its non-hydrolyzable homolog ADPNP) and also of its double mutants D56A + D274A and D135A + D198A complexed to AMP and ADP, respectively. These data reveal strong conservation of the structural features seen in the γ-subunit of AMPK providing insight into the key residues involved in nucleotide binding in the four canonical binding sites. More interestingly, cocrystallization of MJ1225 with AMP, ADP, and ATP revealed a novel AMP binding site that had not been described in CBS proteins so far. This new site is located in a cavity existing between three α-helices that disrupt the internal symmetry of each Bateman domain. A detailed comparison of MJ1225 with its yeast and mammalian homologs revealed that a cavity with similar features can also be recognized in the eukaryal γ-AMPK.

Section snippets

Structure determination and general structural features

The crystal structure of wild-type MJ1225 (herein denoted as MJ1225wt) and of two mutants, D56A + D274A and D135A + D198A (denoted as MJ1225m14 and MJ1225m23), has been determined at 2.1 Å, 2.0 Å, and 2.3 Å, respectively (Fig. 2; Table 1). The three species were recombinantly expressed in Escherichia coli, purified to homogeneity, and crystallized (see Materials and Methods).32 All crystals belong to trigonal space group H32, and analysis based on the Matthews coefficient33 indicated one (VM = 2.7)

Discussion

The strong conservation of the mechanism of nucleotide binding observed in some archaeal proteins has recently led some authors to suggest that a sensor of the energy status similar to that observed in eukaryotes and represented by the AMPK could also exist in prokaryotes.31 The genome of M. jannaschii encodes two stand-alone proteins (MJ1225 and MJ1404) that contain four CBS repeats of similar length arranged as two Bateman domains that probably adopt a head-to-head orientation, as observed in

Cloning, mutagenesis, and purification

MJ1225wt construct (residues 1 to 280) was produced using the protocol described elsewhere.32 The PCR product was cloned in pET101D plasmid using the Champion pET Directional TOPO Expression Kit (Invitrogen). MJ1225 mutant expression constructs were generated in a PCR-based site-directed mutagenesis using the QuikChange mutagenesis kit (Stratagene). First, the single mutants D56A, D135A, D198A, and D274A (denoted here as MJ1225m1, MJ1225m2, MJ1225m3, and MJ1225m5) were generated using as

Acknowledgements

We deeply thank both reviewers for helpful comments, insights, and suggestions. We also thank Adriana Rojas for maintenance of the in-house equipment and the staff of ESRF beamlines ID14-2, ID14-4, ID23-1, and ID23-2 for support during synchrotron data collection. This research was funded by program grants from the Basque Government (ETORTEK IE05-147 and IE07-202), Diputación Foral de Bizkaia (Exp. 7/13/08/2006/11 and 7/13/08/2005/14), the Spanish Ministry of Education (SAF2005-00855), and SICI

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