Structure of a Mutant Adenylate Kinase Ligated with an ATP-analogue Showing Domain Closure Over ATP

103

113

Large conformational changes in the LID and NMP domains of adenylate kinase (AKE) are known to be key to ligand binding and catalysis, yet the order of binding events and domain motion is not well understood. Combining the multiple available structures for AKE with the energy landscape theory for protein folding, a theoretical model was developed for allostery, order of binding events, and efficient catalysis. Coarse-grained models and nonlinear normal mode analysis were used to infer that intrinsic structural fluctuations dominate LID motion, whereas ligand-protein interactions and cracking (local unfolding) are more important during NMP motion. In addition, LID-NMP domain interactions are indispensable for efficient catalysis. LID domain motion precedes NMP domain motion, during both opening and closing. These findings provide a mechanistic explanation for the observed 1:1:1 correspondence between LID domain closure, NMP domain closure, and substrate turnover. This catalytic cycle has likely evolved to reduce misligation, and thus inhibition, of AKE. The separation of allosteric motion into intrinsic structural fluctuations and ligand-induced contributions can be generalized to further our understanding of allosteric transitions in other proteins.Kinase-mediated phosphoryl transfer is a key component of many signaling pathways. Tight control of signaling requires regulation of kinase activity, which is influenced by allosteric transitions (1, 2). The classical description of allostery involves ligand-induced conformational rearrangements between static protein structures. It is now acknowledged that protein dynamics is statistical in nature and that allostery is often due to a change in the balance of pre-existing conformational substates upon ligand binding (3).This manuscript explores the subject of how protein structure determines allostery, order of ligand binding events, and ultimately, efficient catalysis, in the context of adenylate kinase (AKE).4 AKE is a three-domain (LID, NMP, and CORE) protein (Fig. 1) that undergoes large conformational changes as it catalyzes Reaction 1.Large motions of the LID and NMP domains are associated with nucleotide binding. It has been shown that substrate turnover and domain rearrangements ("open" state to "closed" state) occur at the same frequency (2). Despite several theoretical (6 -13) and experimental studies (2, 14 -17) on AKE, a catalytic mechanism that explains this high efficiency has not been proposed.Because it is known that conformational transitions can be well described as a superposition of normal modes (6, 7, 13, 18 -20), we use a simplified nonlinear elastic network model and a structure-based model with implicit ligand interactions to demonstrate a likely catalytic mechanism in AKE. We show that intrinsic structural fluctuations (21,22) in the LID domain account for the majority of the domain's motion and that ATP binding likely serves to lock the domain closed. The LID motion is an example of allosteric regulation that results from a shift in the popu...

Section: Discussionmentioning

confidence: 99%

Conformational Transitions in Adenylate Kinase

Whitford¹,

Gosavi²,

Onuchic

2008

103

113

“…However, a mechanism similar to that of adenylate kinase (code 1AKY) involving the attack of a catalytic water molecule cannot be discounted. Mutation of the corresponding Asp residue of adenylate kinase results in loss of NTP binding (36).…”

Section: Discussionmentioning

confidence: 99%

Juvenile Hormone Diol Kinase

Maxwell¹,

Welch²,

Horodyski³

et al. 2002

The gene sequence of Manduca sexta juvenile hormone diol kinase (JHDK) codes for an enzyme that has 59% sequence identity to Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2). JHDK and dSCP2 are similar to G-proteins with three conserved sequence elements involved in purine nucleotide binding. Both proteins contain two pairs of EF-hand motifs. Characterization and partial purification of the D. melanogaster homolog of M. sexta JHDK from adult D. melanogaster gave material with JHDK activity. This activity has an experimental pI and molecular mass that are nearly identical to those of dSCP2. Moreover, D. melanogaster phosphotransferase activity has very similar chromatographic retention in three systems compared with M. sexta JHDK. Substrate docking to threedimensional models of JHDK has shown that the three conserved nucleotide-binding elements surround the putative substrate-binding site and align with conserved sequence elements of p21Ras and adenylate kinase. D. melanogaster dSCP2 is a homolog of M. sexta JHDK, and these proteins constitute a novel kinase family that binds nucleotides using the scaffold of an SCP (Protein Data Bank code 2SCP).Endocrine control of embryonic and post-embryonic development in insects is primarily accomplished by juvenile hormone (JH) 1 and ecdysteroids (1). Development proceeds through a series of molts and metamorphosis, where the function of JH is to regulate the character of the molt; metamorphosis is characterized by a sharp decrease in JH, and adult molting by the absence of JH (1). JH levels are regulated by de novo biosynthesis (2) and catabolism (3). Cellular JH metabolism involves a microsomal JH epoxide hydrolase that metabolizes JH to JH diol and a soluble JH diol kinase (JHDK) that catalyzes the formation of JH diol phosphate. JHDK is a homodimer with a subunit molecular mass of 20 kDa that uses MgATP or MgGTP as a phosphate donor to effectively catabolize JH I, II, or III diol by placing a phosphate group on the C-10 hydroxyl, thereby creating JH diol phosphate, the water-soluble end metabolite of JH catabolism (4).In this study, we report the sequencing and cloning of Manduca sexta JHDK, the molecular modeling of JHDK and Drosophila melanogaster sarcoplasmic calcium-binding protein-2 (dSCP2), and the partial purification and characterization of the D. melanogaster homolog of M. sexta JHDK. We show with high probability that the product of the D. melanogaster dSCP2 gene is the D. melanogaster homolog of M. sexta JHDK. Each is a member of a novel kinase family that is structurally similar to SCPs. EXPERIMENTAL PROCEDURES Chemicals and D. melanogaster Protein Preparations-Ampholyteswere obtained from Bio-Rad. 3-Octylthio-1,1,1-trifluoro-2-propanone was from Dr. Bruce D. Hammock (University of California, Davis, CA). Aprotinin, 4-(2-aminoethyl)benzenesulfonyl fluoride, pepstatin, and high-grade (NH 4 ) 2 SO 4 were purchased from Sigma. Lysyl endopeptidase (Lys-C) was obtained from Wako Bioproducts (Richmond, VA). The synthesis of (10S,11S)- [10-3 H...

“…The enzyme contains two distinct nucleotide binding sites: the MgATP site, which binds MgATP and MgADP, and the AMP site, which is specific for AMP and uncomplexed ADP. The substrate-induced conformation changes in AK have been the subject of a number of investigations (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Based on the comparison of AK crystal structures representing the enzyme in different ligand forms, apo-form (from pig muscle), enzyme-AMP binary complex (from beef heart mitochondrial matrix), and enzyme-AP 5 A complex (from Escherichia coli), Schulz and co-workers (15,18) suggested that AK undergoes large structural changes upon substrates binding.…”

mentioning

confidence: 99%

Evidence for at Least Two Native Forms of Rabbit Muscle Adenylate Kinase in Equilibrium in Aqueous Solution

Zhang

Sheng

Niu

et al. 1998

The time course of 8-anilino-1-naphthalenesulfonic acid (ANS) binding to adenylate kinase (AK) is a biphasic process. The burst phase ends in the dead-time of the stopped-flow apparatus (about 15 ms), whereas the slow phase completes in about 10 min. A Job's plot tests of the binding stoichiometry demonstrates that there is one ANS binding site on AK, but only about 70% of the enzyme can rapidly bind with ANS, indicating that the conformation of native AK molecules is not homogeneous. Further kinetic analysis shows that the effects of ANS and substrates concentration on the burst and slow phase fluorescence building agree well with the multiple native forms mechanism. One form (denoted N 1 ) binds with ANS, whereas the other (denoted N 2 ) does not. ANS binding to N 1 results in a burst phase fluorescence increase, followed by the interconversion of N 2 to N 1 , to give the slow phase ANS binding. Under urea denaturation conditions, N 2 is easily perturbed by urea and unfolds completely at low denaturant concentrations, whereas N 1 is relatively resistant to denaturation and unfolds at higher denaturant concentrations. The existence of multiple native forms in solution may shed some light on the interpretation of the enzyme catalytic mechanism.It has been accepted that a globular protein in its native state adopts a single, well defined conformation. However, this concept has been challenged by several reports that some proteins may exist in more than one distinct folded form in equilibrium. Evidence for distinguishing multiple native forms of staphylococcal nuclease has come from electrophoretic and NMR studies (1-6). For calbindin D 9K , evidence of multiple forms came not only from NMR studies, but also from x-ray crystal structure (7,8).Adenylate kinase (AK 1 ; EC 2.7.4.3) catalyzes the phosphoryl transfer reaction: MgATP ϩ AMP i MgADP ϩ ADP (9, 10). The enzyme contains two distinct nucleotide binding sites: the MgATP site, which binds MgATP and MgADP, and the AMP site, which is specific for AMP and uncomplexed ADP. The substrate-induced conformation changes in AK have been the subject of a number of investigations (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). Based on the comparison of AK crystal structures representing the enzyme in different ligand forms, apo-form (from pig muscle), enzyme-AMP binary complex (from beef heart mitochondrial matrix), and enzyme-AP 5 A complex (from Escherichia coli), Schulz and co-workers (15, 18) suggested that AK undergoes large structural changes upon substrates binding. These conformational changes can be subdivided into two steps; the first change, corresponding to binding to AMP, only involves the displacement of the small ␣-helical domain (residues 30 -59 in E. coli AK), whereas the second change, occurring with additional binding of substrate ATP, mainly involves the displacement of the LID domain (residues 122-159 in E. coli AK). However, it is not clear whether the enzyme achieves its catalytic conformation only upon substrate binding or whether the above confo...