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

Abstract

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 ...

Hotspots in an Obligate Homodimeric Anticancer Target. Structural and Functional Effects of Interfacial Mutations in Human Thymidylate Synthase

“…As previously observed for the WT enzyme when crystallized in the inactive conformation (PDB code 3NG5), 2 two sulfate ions are bound at the dimer interface in the mutants, labeled site 1 and site 2 in Figure 5. The positions and coordination of these sulfate ions are not significantly affected by the mutations.…”

“…In a previous study, we identified seven peptides designed from the dimer interface that inhibit the catalytic activity of the protein by binding at the monomer–monomer interface of the di-inactive form of the protein. 2 The interaction of these peptides with four dimer interface mutants, K47A, F59A, L198A, and Y202A, was explored by Tochowicz et al 28 The results of the present hot-spot structural and functional analysis are here analyzed in combination with those of the above peptide inhibitor studies.…”

“…5 In the inactive form, the loop twists so that instead of pointing into the active site, the catalytic C195 points toward the dimer interface. Cardinale et al 2 identified peptides that bind at the dimer interface of hTS and thereby stabilize the inactive conformation of the protein. It has been shown that the TS dimer can be denatured into unfolded monomers and refolded to its native state.…”

Bacterial Thymidylate Synthase Binds Two Molecules of Substrate and Cofactor without Cooperativity

“…We are currently investigating this coupling. Lastly, we should emphasize that our work does not necessarily reflect on reports that eukaryotic TSase is a cooperative enzyme because, unlike the enzyme studied here, symmetry is broken in forms from higher organisms by an active site appendage that adopts multiple conformations in the absence of ligands 32,33 .…”

Targeting protein dynamics in drug design

“…I will first discuss some of the different ways in which protein dynamics can be targeted, giving examples from our experience in the design of inhibitors of thymidylate synthase [1-3]. I will then describe the development of a computational toolbox (TRAPP: TRAnsient Pockets in Proteins) to identify and visualize transient pockets in proteins that may be exploited in ligand design.…”