Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes

Błaszczyk, Jarosław; Li, Yue; Yan, Honggao; Ji, Xinhua

doi:10.1006/jmbi.2000.4427

Cited by 78 publications

(120 citation statements)

References 41 publications

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“…Overall Structure-The overall fold of the mouse guanylate kinase is very similar to that of the yeast enzyme (6). Consisting of 198 amino acid residues, mGMPK is 11 residues longer than yGMPK; two of these amino acids are located at the N terminus, and nine are located at the C-terminal part of the protein.…”

Section: Resultsmentioning

confidence: 99%

“…The mGMPK in complex with GMP and ADP is in a closed conformation, which allows us, by comparing it with previously reported open (yGMPK apo ) and partially closed (yGMPK GMP ) structures of yGMPK (6), to delineate the effect of each nucleotide on the conformation of the enzyme (Fig. 3).…”

Section: Fig 1 Ribbon Diagram Of Mgmpk Gmp-adpmentioning

confidence: 95%

“…Therefore, it is medically important to elucidate its enzymatic mechanism and the structural basis for its nucleotide specificity. Our current structural understanding of this enzyme is derived from the apo-and GMP-bound structures of the yeast GMPK (6) and from analogy to other nucleoside monophosphate (NMP) kinases (7).…”

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confidence: 99%

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Structural Characterization of the Closed Conformation of Mouse Guanylate Kinase

Sekulić¹,

Shuvalova²,

Spangenberg³

et al. 2002

Journal of Biological Chemistry

121

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Guanylate kinase (GMPK) is a nucleoside monophosphate kinase that catalyzes the reversible phosphoryl transfer from ATP to GMP to yield ADP and GDP. In addition to phosphorylating GMP, antiviral prodrugs such as acyclovir, ganciclovir, and carbovir and anticancer prodrugs such as the thiopurines are dependent on GMPK for their activation. Hence, structural information on mammalian GMPK could play a role in the design of improved antiviral and antineoplastic agents. Here we present the structure of the mouse enzyme in an abortive complex with the nucleotides ADP and GMP, refined at 2.1 Å resolution with a final crystallographic R factor of 0.19 (R free ‫؍‬ 0.23). Guanylate kinase is a member of the nucleoside monophosphate (NMP) kinase family, a family of enzymes that despite having a low primary structure identity share a similar fold, which consists of three structurally distinct regions termed the CORE, LID, and NMP-binding regions. Previous studies on the yeast enzyme have shown that these parts move as rigid bodies upon substrate binding. It has been proposed that consecutive binding of substrates leads to "closing" of the active site bringing the NMP-binding and LID regions closer to each other and to the CORE region. Our structure, which is the first of any guanylate kinase with both substrates bound, supports this hypothesis. It also reveals the binding site of ATP and implicates arginines 44, 137, and 148 (in addition to the invariant P-loop lysine) as candidates for catalyzing the chemical step of the phosphoryl transfer.Guanylate kinase (GMPK, 1 ATP:GMP phosphotransferase, EC 2.7.4.8) is a critical enzyme for the biosynthesis of GTP and dGTP by catalyzing the phosphoryl transfer from ATP to (d)GMP resulting in ADP and (d)GDP (1, 2). GMPK also plays an important role in the recycling of the second messenger cGMP (3). In addition to these physiological roles, GMPK is essential for the activation of prodrugs used for the treatment of cancers and viral infections (4, 5). Therefore, it is medically important to elucidate its enzymatic mechanism and the structural basis for its nucleotide specificity. Our current structural understanding of this enzyme is derived from the apo-and GMP-bound structures of the yeast GMPK (6) and from analogy to other nucleoside monophosphate (NMP) kinases (7).It has been shown that the induced fit mechanism (8) plays an important role in NMP kinases, of which adenylate kinase is the best characterized (9 -11). NMP kinases catalyze phosphoryl transfer by binding both donor and acceptor nucleotides to form a ternary complex. Comparison of the crystal structures of nucleotide-free adenylate kinase to the one in which a single substrate is bound (AMP or ATP) and to the complex in which both substrates are present revealed the conformational changes that occur along the reaction coordinate: from an open unbound enzyme via a partially closed intermediate in which a single substrate is present to the fully closed form in the presence of both substrates. These substrate-induced conform...

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Section: Resultsmentioning

confidence: 99%

Section: Fig 1 Ribbon Diagram Of Mgmpk Gmp-adpmentioning

confidence: 95%

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Structural Characterization of the Closed Conformation of Mouse Guanylate Kinase

Sekulić¹,

Shuvalova²,

Spangenberg³

et al. 2002

Journal of Biological Chemistry

121

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“…The MAGUK GK dom functions as a protein interaction domain and is involved in more sophisticated cellular processes such as stabilizing cell-cell adhesions and orientation of the mitotic spindle (5,7,8). Although GK enz and GK dom have high sequence and structural similarity (9)(10)(11), GK enz has enzymatic activity but no known peptide ligands, whereas GK dom has multiple peptide ligands but no known enzymatic activity (5,12). An understanding of the sequence differences between GK enz and GK dom that support their distinct functions may illuminate the molecular processes that lead to the creation of new protein functions.…”

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confidence: 99%

Conversion of the enzyme guanylate kinase into a mitotic-spindle orienting protein by a single mutation that inhibits GMP-induced closing

Johnston

Whitney

Volkman

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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New protein functions can require complex sequence changes, but the minimal path is not well understood. The guanylate kinase enzyme (GK enz ), which catalyzes phosphotransfer from ATP to GMP, evolved into the GK domain (GK dom ), a protein-binding domain found in membrane associate guanylate kinases that function in mitotic spindle orientation and cell adhesion. Using an induced polarity assay for GK dom function, we show that a single serine to proline mutation is sufficient to switch extant GK enz into a functional GK dom . The mutation blocks catalysis (GK enz function) but allows protein binding and spindle orientation (GK dom function). Furthermore, whereas the GK enz undergoes a large closing motion upon GMP binding, fluorescence quenching and NMR demonstrate that the S → P mutation inhibits GMP-induced GK movements. Disrupting GK closing with a mutation at a different position also leads to GK dom function, suggesting that blocking the GK enz closing motion is sufficient for functional conversion of GK enz to GK dom . Although subtle changes in protein function can require complex sequence paths, our work shows that entirely new functions can arise from single mutations that alter protein dynamics.protein interactions | neofunctionalization | protein engineering | molecular evolution P roteins perform remarkably diverse functions including catalysis, small molecule binding, and protein recognition. Molecular evolution seeks to understand how the diversity of known protein functions arose, whereas the goal of protein engineering is to create new ones (1). An evolutionary process for creating new protein functions is the divergent evolution of duplicated genes, in which gene duplication is followed by functional evolution [neofunctionalization; (2)]. However, loss of function (pseudogene formation or nonfunctionalization) competes with functional evolution and may be much more likely (3). The degree to which neofunctionalization can "win out" over pseudogene formation depends on the number of mutations required to attain the new function and the possible paths in sequence space to the new function. Thus, neofunctionalization is a competitive process that is dependent on the number of required mutations and how protein function is achieved. Insight into the mechanisms of neofunctionalization should improve our ability to develop previously undescribed protein functions.We examined neofunctionalization in the context of a dramatic functional change: creation of a mitotic spindle-orienting protein from a nucleotide kinase within the membrane associated guanylate kinase (MAGUK) family of proteins. MAGUKs are proteins that mediate intricate multicellular functions such as cell junction formation and mitotic spindle orientation and have been found only in metazoans and choanoflagellates (4, 5).

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“…The protein is attached to the gold surfaces through Cysteines introduced by mutagenesis on the two lobes of the structure. The "hinge motion" [11] which transforms the structure from the open to the closed form (Fig. 2) upon binding the substrate GMP -a classic example of induced fit [1] -roughly corresponds to moving the two Cysteines by 1 nm towards each other.…”

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confidence: 99%

Cracking phase diagram for the dynamics of an enzyme

Landy

Zocchi

2012

Phys. Rev. E

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We measure the ensemble averaged deformation of an enzyme for an oscillating applied force. From the low frequency divergence of the mechanical susceptibility for the hinge motion of Guanylate Kinase we obtain a non-equilibrium phase diagram in the frequency -force plane. A phase line separates linear elasticity dynamics from softer (viscoelastic) dynamics. The hinge motion corresponds to crossing this phase line (not to a soft linear elastic mode). The phase line is dramatically shifted in the closed state compared to the open state of the enzyme.Introduction. Virtually all enzymes couple catalysis to conformational motion. Evident in motor proteins, mechano-chemical coupling is also the basis for substrate specificity [1,2] and activity regulation [3][4][5] in these molecular machines. Conformational changes coupled to catalysis are often large, with amplitudes of ∼ 1 nm compared to an overall size of the enzyme of ∼ 5 nm. The folded protein being a solid, these huge strains pose an interesting question about the nature of conformational dynamics and the associated materials properties of the system. Surely this motion is not within the linear elasticity regime, and a specific nonlinear mechanism for these transitions, called "cracking", was proposed some years ago [6,7]. Cracking connects initial and final states through a local melting and refolding event. Translated into stress-strain relations, this should give rise to an interesting dynamics. Here we measure directly the stress-strain relations for the hinge motion of the enzyme Guanylate Kinase (GK), a molecule quite similar to the enzyme considered in [6,7]. Specifically, we report comparative nano-rheology [8] measurements on the open (no substrate) and closed (with the substrate GMP) forms of the enzyme and find that while the linear elasticity regime is almost the same, the nonlinear behavior, or what we have called the viscoelastic regime [9,10] is dramatically different in the two cases. A phase diagram in the frequency -force plane maps out regions of linear elastic vs softer (viscoelastic) dynamics. The existence of this phase line may be a universal feature of the dynamics of enzymes.

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Crystal structure of unligated guanylate kinase from yeast reveals GMP-induced conformational changes

Cited by 78 publications

References 41 publications

Structural Characterization of the Closed Conformation of Mouse Guanylate Kinase

Structural Characterization of the Closed Conformation of Mouse Guanylate Kinase

Conversion of the enzyme guanylate kinase into a mitotic-spindle orienting protein by a single mutation that inhibits GMP-induced closing

Cracking phase diagram for the dynamics of an enzyme

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