Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors<sup>,</sup>

Lovelace, L.L.; Gibson, Lydia M.; Lebioda, Lukasz

doi:10.1021/bi061309j

Cited by 34 publications

(50 citation statements)

References 34 publications

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“…Therefore, it is important to identify hTS inhibitors that act through new mechanisms that do not alter RNA regulation or increase protein levels. With this aim, some diphosphonic acids have been proposed as allosteric inhibitors of hTS (33). The most active one is 1,3-propanediphosphonic acid (PDPA) which binds at the position where the phosphate group of dUMP binds.…”

mentioning

confidence: 99%

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Cardinale

Guaitoli

Tondi

et al. 2011

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

157

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The undersigned authors note the following: "We wish to bring to your attention an issue regarding our PNAS publication referenced above. Although we cite our earlier PNAS publication (see ref. Figs. 2 and 3 display the UWHBs for Hb β-subunit (pdb.1bz0, chain B) and human cellular prion protein (pdb.1qm0) (12)(13)(14). Within the natural interactive context of the Hb subunit, the UWHBs signal crucial binding regions (24): UWHBs (90, 94), (90, 95) are associated with the β-FG corner involved in the quaternary α1β2 interface; UWHB (5, 9) is adjacent to Glu-6 which in sickle cell anemia mutates to Val-6 and is located at the Val-6-(Phe-85, Leu-88) interface in the deoxyHbS fiber."The following text in the section titled 'Toward a Structural Diagnosis' on page 6449 of our text is similar to the text beginning in the last paragraph on page 2392 in ref. 23:The distribution of proteins according to their average extent of hydrogen bond wrapping and their spatial concentration of structural defects is shown in Fig. 5 (see also ref. 23). The sample of 2,811 PDB proteins is large enough to define a reliable abundance distribution with an inflection point at ρ = 6.20. The integration of the distribution over a ρ-interval gives the fraction of proteins whose ρ lies within that range. Of the 2,811 proteins examined, 2,572 have ρ > 6.20, and none of them is known to yield amyloid aggregation under physiological conditions entailing partial retention of structure. Strikingly, relatively few disease-related amyloidogenic proteins are known in the sparsely populated, underwrapped 3.5 < ρ < 6.20 range, with the cellular prion proteins located at the extreme of the spectrum (3.53 < ρ < 3.72)....The range of H-bond wrapping 3.5 < ρ < 4.6 of 20 sampled PDB membrane proteins has been included in Fig. 5 for comparison. As expected, such proteins do not have the stringent H-bond packing requirements of soluble proteins for their H bonds at the lipid interface. Thus, this comparison becomes suggestive in terms of elucidating the driving factor for aggregation in soluble proteins: Although the UWHB constitutes a structural defect in a soluble protein because of its vulnerability to water attack, it is not a structural defect in a membrane protein. The exposure of the polar amide and carbonyl of the unbound state to a nonpolar phase is thermodynamically unfavorable (22). The virtually identical ρ value for human prion and outer-membrane protein A (Fig. 5) is revealing in this regard.Furthermore, all known amyloidogenic proteins that occur naturally in complexed form have sufficient H-bond wrapping within their respective complexes (ρ value near 6.2). Their amyloidogenic propensity appears only under conditions in which the protein is dissociated from the complex (compare Fig. 5). This finding is corroborated by the following computation. If an intramolecular hydrogen bond is underwrapped within the isolated protein molecule but located at an interface upon complexation, then to determine its extent of wrapping within the complex, we take ...

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mentioning

confidence: 99%

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Cardinale

Guaitoli

Tondi

et al. 2011

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

157

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“…Although dUMP was recently shown to bind with minimal cooperativity for the E. coli enzyme at 25°C, there are signs of unequal thermodynamics between the two protomers at lower temperatures (16), and indeed data herein show clear intersubunit communication. Moreover, other TS enzymes, and in particular human, seem to show more dramatic cooperativity, suggesting that intersubunit communication is an intrinsic feature of TS (13,(17)(18)(19)(20)(21). To overcome the difficulties of studying symmetric proteins by NMR, we generated a pair of mixed labeled dimers of TS that each have a single functional active site and a single protomer labeled for NMR studies.…”

Section: Significancementioning

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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“…Trp fluorescence was used to observe the antifolate binding regarding to the Trp residues at the active site (W80 and W83) (Anderson, O'Neil, DeLano & Stroud, 1999;Felder, Dunlap, Dix & Spencer, 2002;Lovelace, Gibson & Lebioda, 2007;Sharma & Kisliuk, 1975). Samples were prepared by dialysis as described above for ITC.…”

Section: Tryptophan Fluorescencementioning

confidence: 99%

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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Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors^,

Cited by 34 publications

References 34 publications

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Chemical shift imprint of intersubunit communication in a symmetric homodimer

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

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Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors,

Cited by 34 publications

References 34 publications

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Protein–protein interface-binding peptides inhibit the cancer therapy target human thymidylate synthase

Chemical shift imprint of intersubunit communication in a symmetric homodimer

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

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Cooperative Inhibition of Human Thymidylate Synthase by Mixtures of Active Site Binding and Allosteric Inhibitors^,