NMR-Guided Structural Elucidation of c-kit2 G-Quadruplexes

The structures of the ckit2 G-quadruplexes were calculated using the X-PLOR (30 ) and XPLOR-NIH (v.2.11-2) (31 (link)) programs as described previously (32 (link)), with protocols differing in order to account for non-crystallographic symmetry of the dimeric form-II G-quadruplex. The initial folds guided by NMR restraints listed in Tables 1 and 2 were obtained using torsion dynamics with R⁻⁶ distance averaging for monomeric form-I and sum-averaging (with ambiguous restraints) for dimeric form-II G-quadruplexes. The distance restraints for dimeric form-II G-quadruplex obtained from build-up measurements were augmented by single-mixing time (300 ms) distances from the NOESY spectrum of the I4 analog of c-kit2 promoter sequence (which showed better spectral resolution of resonances associated with G3 and I4 residues). The structures were further refined by Cartesian dynamics and, finally, using relaxation matrix refinement.
Table 1.

Statistics of NMR restraint-guided computations of c-kit2 promoter monomeric form-I G- quadruplex

A. NMR restraints
Distance restraintsa	Non-exchangeable		Exchangeable
Intra-residue distance restraints	158		0
Sequential (i, i+1) distance restraints	61		10
Long-range (i, ≥ i+2) distance restraints	7		29
Other restraints
Hydrogen bonding restraints
(H-N, H-O, and heavy atoms)		52
Torsion angle restraintsa		54
Intensity restraints
Non-exchangeable protons (each of four mixing times		223
B. Structure statistics of 12 molecules following intensity refinement
NOE violations
Number (>0.2 Å)		0.25 ± 0.45
r.m.s.d. of violations		0.02 ± 0.00
Deviations from the ideal covalent geometry
Bond lengths (Å)		0.01 ± 0.00
Bond angles (deg.)		0.86 ± 0.02
Impropers (deg)		0.42 ± 0.03
NMR R-factor (R1/6)		0.03 ± 0.01
Pairwise all heavy atom r.m.s.d. values (12 refined structures)
All heavy atoms in G-tetrads		0.43 ± 0.09
All heavy atoms except C9-T12		0.62 ± 0.12
All heavy atoms		1.49 ± 0.51

^aAll residues were restrained to χ values in the 240 (±70)° range, characteristic of anti glycosidic torsion values.

The ε of the residues C1-G20 was restrained to the stereochemically allowed range 225 (±75)°. The γ torsion angle of the residues 2–4, 6–8, 14–16 and 18–21 was restrained to the values of 60 (±35)° identified experimentally.

Table 2.

Statistics of NMR restraint-guided computations of c-kit2 promoter dimeric form-II G-quadruplex

A. NMR restraints
Distance restraintsa	Non-exchangeable		Exchangeable
Intra-residue distance restraints	189		0
Sequential (i, i+1) distance restraints	87		11
Long-range (i, ≥ i+2) distance restraints	6		42
Other restraints
Hydrogen bonding restraints
(H-N, H-O, and heavy atoms)		104
Torsion angle restraintsa		155
Intensity restraints
Non-exchangeable protons (each of four mixing times)		145
B. Structure statistics of 10 molecules following intensity refinement
NOE violations
Number (>0.2 Å)		0.40 ± 0.70
r.m.s.d. of violations		0.03 ± 0.00
Deviations from the ideal covalent geometry
Bond lengths (Å)		0.06 ± 0.00
Bond angles (deg.)		0.79 ± 0.06
Impropers (deg)		0.45 ± 0.03
NMR R-factor (R1/6)		0.03 ± 0.00
Pairwise all heavy atom r.m.s.d. values (10 refined structures)
All heavy atoms in G-tetrads		0.57 ± 0.17
All heavy atoms except C5, A17		0.79 ± 0.26
All heavy atoms		1.23 ± 0.29

^aAll residues were restrained to χ values in the 240 (±70)° range, characteristic of anti glycosidic torsion values.

The ε of the residues C1-G20 was restrained to the stereochemically allowed range 225 (±75)°.

The γ torsion angle of the residues 1–4, 7–8, 14–16 and 19–21 was restrained to the values of 60 (±35)°, the sugar pucker of the residues 2–4, 6–8, 14–16 and 18–20 was restrained in C2′-endo domain, identified experimentally.

The initial fold consisted of an extended DNA strand (two strands in the case of form-II) with randomized chain torsion angles of constituent nucleotides, whose angles and bonds were set up in accordance with the most updated measurements (33 ,34 ). Folding of the dimeric form-II G-quadruplex from two extended strands resulted in substantial overpopulation by high-energetic left-handed forms, with the stacking order inverted relative to the chemical order of bases. The initial folding of the dimeric form-II G-quadruplex was therefore achieved in two steps. In the first step, only restraints for the 5′-end G-quadruplex were activated. Three independently obtained 5′-end associated right-handed G-quadruplex molecules were used in the second step, where restraints for both 5′-end and 3′-end G-quadruplexes were activated. At initial stages of dimeric form-II G-quadruplex computations, with lesser amount of restraints used, some 3′- to 3′-end oriented G-quadruplex associations were obtained (with five-residue linker in extended conformation). The NMR spectra were examined for the possible formation of cross-peaks indicating presence of dimeric 3′- to 3′-end G-quadruplex association. We do not observe NOE cross-peaks between the methyl group of T21 and imino-protons of G4 and G8, which, together with other set of observed and assigned cross-peaks, rule out formation of a dimeric 3′- to 3′-end (tail-to-tail) G-quadruplex, in favor of a dimeric 3′- to 5′-end (tail-to-head) G-quadruplex.