Use of Strain in a Stereospecific Catalytic Mechanism:  Crystal Structures of <i>Escherichia coli</i> Thymidylate Synthase Bound to FdUMP and Methylenetetrahydrofolate<sup>,</sup>

Hyatt, David C.; Maley, Frank; Montfort, W.R.

doi:10.1021/bi962936j

Cited by 78 publications

(131 citation statements)

References 39 publications

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“…The most abundant one is the bent conformation, as found in dihydrofolate reductase and other enzymes (32). In crystal structures of enzymes with 5-CH 3 -H 4 PteGlu and with 5-CH 3 -H 4 PteGlu 5 , some contain folate in the bent conformation, whereas others contain folate in the extended form (32)(33)(34)(35). The best assignment of the folate in the electron density in the GNMT-folate complex was achieved when the folate was built in almost extended conformation as shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

Luka

Pakhomova

Loukachevitch

et al. 2007

Journal of Biological Chemistry

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Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. It is abundant in the liver, where it uses excess S-adenosylmethionine (AdoMet) to methylate glycine to N-methylglycine (sarcosine) and produces S-adenosylhomocysteine (AdoHcy), thereby controlling the methylating potential of the cell. GNMT also links utilization of preformed methyl groups, in the form of methionine, to their de novo synthesis, because it is inhibited by a specific form of folate, 5-methyltetrahydrofolate. Although the structure of the enzyme has been elucidated by x-ray crystallography of the apoenzyme and in the presence of the substrate, the location of the folate inhibitor in the tetrameric structure has not been identified. We report here for the first time the crystal structure of rat GNMT complexed with 5-methyltetrahydrofolate. In the GNMT-folate complex, two folate binding sites were located in the intersubunit areas of the tetramer. Each folate binding site is formed primarily by two 1-7 N-terminal regions of one pair of subunits and two 205-218 regions of the other pair of subunits. Both the pteridine and p-aminobenzoyl rings are located in the hydrophobic cavities formed by Tyr 5 , Leu 207 , and Met 215 residues of all subunits. Binding experiments in solution also confirm that one GNMT tetramer binds two folate molecules. For the enzymatic reaction to take place, the N-terminal fragments of GNMT must have a significant degree of conformational freedom to provide access to the active sites. The presence of the folate in this position provides a mechanism for its inhibition.

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Section: Resultsmentioning

confidence: 99%

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

Luka

Pakhomova

Loukachevitch

et al. 2007

Journal of Biological Chemistry

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“…2). This interaction was observed in the crystal structure of the wild-type (WT) ternary covalent complex of TS with FdUMP and CH 2 THF (Hyatt, Maley & Montfort, 1997) indicating that K48 is essential for the binding of CH 2 THF and its analogues (Kamb, Finer-Moore, Calvert & Stroud, 1992). The CH 2 THF γ-glutamate is critical for folate 1 K48 is referred to the E. coli sequence numbering and is equivalent to K77 in the human TS amino acid sequence.…”

Section: Introductionmentioning

confidence: 91%

“…Since in the latter structure, K48 contacts the main chain of I258 through a water molecule, while Y4 and Q219 are also H-bonded through a water molecule (Hyatt, Maley & Montfort, 1997). However, in the K48Q mutant binary complex, the contact with the F171 carbonyl is lost and the side-chain carbonyl of Q48 now contacts the main chain amide of I258 (Fig.…”

Section: Nucleotide Binding To the K48q Mutantmentioning

confidence: 98%

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Arvizu‐Flores

Sugich-Miranda

Arreola

et al. 2008

The International Journal of Biochemistry & Cell Biology

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Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH 2 THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k cat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the K m for the (R)-stereoisomer of CH 2 THF was 300 µM, about 30 fold larger than K m from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH 2 THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

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“…Although the mixed dimers are required to visualize the dUMP 1 states, they are not required for the cofactor binding step of the reaction. Binding of a substrate analog, 5-FdUMP, along with cofactor, together referred to as the "diligand," forms a covalent bond to the enzyme, leading to a stable ternary complex (22,23). Because of the covalent nature of this complex, the resonances from the diligandbound TS are in slow exchange, and, thus, the chemical shifts of the diligand 1 states can be more easily measured (SI Materials and Methods) (16).…”

mentioning

confidence: 99%

“…Because of the covalent nature of this complex, the resonances from the diligandbound TS are in slow exchange, and, thus, the chemical shifts of the diligand 1 states can be more easily measured (SI Materials and Methods) (16). Together, dUMP and diligand will allow us to compare the proteins' response, not only to an additional binding event but also to a conformational change, because, unlike dUMP binding, diligand binding causes significant conformational changes in the vicinity of the binding site to form the closed ternary complex (22,24).…”

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confidence: 99%

Chemical shift imprint of intersubunit communication in a symmetric homodimer

Falk

Sapienza

Lee

2016

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

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Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein-ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state ("lig 1 ") of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig 1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig 1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP 1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP 1 peaks are found beyond the limits set by apo and dUMP 2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers.A llosteric regulation in proteins is a ubiquitous mechanism for controlling cellular behavior and an attractive strategy for therapeutic development. Even though broadly recognized, longrange communication is not well understood mechanistically (1-4). Although there have been numerous strategies to reveal the structural and dynamic underpinnings of allostery, oddly these strategies have largely focused on complex oligomeric or, alternatively, on small monomeric allosteric proteins. A likely more straightforward approach is to study allosteric mechanisms using simple symmetric homodimeric proteins, which would allow for answering the basic and general question of how the occurrence of an event in one subunit is communicated to another subunit, as occurs in classical multisubunit allosteric proteins. Given the large number of homodimeric proteins involved in cellular regulation (such as growth factors, cytokines, kinases, G protein-coupled receptors, transcription factors, and metabolic proteins), insights into intersubunit communication should be widely beneficial (5).As a key step toward a broad understanding of allosteric mechanisms, it will be important to observe how binding a ligand in one subunit is communicated to a second subunit, even in the absence of conformational change. Although this communication is straightforwar...

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Use of Strain in a Stereospecific Catalytic Mechanism: Crystal Structures of Escherichia coli Thymidylate Synthase Bound to FdUMP and Methylenetetrahydrofolate^,

Cited by 78 publications

References 39 publications

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Chemical shift imprint of intersubunit communication in a symmetric homodimer

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Use of Strain in a Stereospecific Catalytic Mechanism: Crystal Structures of Escherichia coli Thymidylate Synthase Bound to FdUMP and Methylenetetrahydrofolate,

Cited by 78 publications

References 39 publications

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

5-Methyltetrahydrofolate Is Bound in Intersubunit Areas of Rat Liver Folate-binding Protein Glycine N-Methyltransferase

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: Calorimetric and crystallographic analysis of the K48Q mutant

Chemical shift imprint of intersubunit communication in a symmetric homodimer

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Use of Strain in a Stereospecific Catalytic Mechanism: Crystal Structures of Escherichia coli Thymidylate Synthase Bound to FdUMP and Methylenetetrahydrofolate^,