Structure and Mechanism of the Influenza A M218-60 Dimer of Dimers

Abstract

We report a magic angle spinning (MAS) NMR structure of the drug-resistant S31N mutation of M218-60 from Influenza A. The protein was dispersed in diphytanoyl-sn-glycero-3-phosphocholine lipid bilayers, and the spectra and an extensive set of constraints indicate that M218-60 consists of a dimer of dimers. In particular, ∼280 structural constraints were obtained using dipole recoupling experiments that yielded well-resolved (13)C-(15)N, (13)C-(13)C, and (1)H-(15)N 2D, 3D, and 4D MAS spectra, all of which show cross-peak doubling. Interhelical distances were measured using mixed (15)N/(13)C labeling and with deuterated protein, MAS at ωr/2π = 60 kHz, ω0H/2π = 1000 MHz, and (1)H detection of methyl-methyl contacts. The experiments reveal a compact structure consisting of a tetramer composed of four transmembrane helices, in which two opposing helices are displaced and rotated in the direction of the membrane normal relative to a four-fold symmetric arrangement, yielding a two-fold symmetric structure. Side chain conformations of the important gating and pH-sensing residues W41 and H37 are found to differ markedly from four-fold symmetry. The rmsd of the structure is 0.7 Å for backbone heavy atoms and 1.1 Å for all heavy atoms. This two-fold symmetric structure is different from all of the previous structures of M2, many of which were determined in detergent and/or with shorter constructs that are not fully active. The structure has implications for the mechanism of H(+) transport since the distance between His and Trp residues on different helices is found to be short. The structure also exhibits two-fold symmetry in the vicinity of the binding site of adamantyl inhibitors, and steric constraints may explain the mechanism of the drug-resistant S31N mutation.

Figure 1

¹⁵N–¹³Cα region of the ZF-TEDOR spectrum of U-¹³C,¹⁵N S31N M2; τ_mix = 1.2 ms was used in order to observe only one bond transfer. Note the two sets of cross-peaks (labeled with blue and black) that correspond to the two different molecules of the M2 dimer; ω_0H/ 2π = 900 MHz.

Figure 2

Selected strips from the 3D ZF-TEDOR-RFDR spectrum recorded at ω_0H/2π = 900 MHz, showing the consecutive assignment of residues P25 to A29. Mixing periods were τ_mix = 1.2 and 4.8 ms for ZF-TEDOR and RFDR, respectively, at ω_r/2π = 20 kHz. The NCACX transfer is shown in red and NCOCX transfer in blue.

Figure 3

Cross-polarization-based HN spectrum showing the completed backbone amide assignments for M2_18–60. Note the cross-peak doubling denoted with blue and black labels on the cross-peaks in the spectrum; ω_r/2π = 60 kHz and ω_0H/2π = 1000 MHz.

Figure 4

Selected strips from the 3D (H)C_αNH spectrum of ²H M2_18–60 back exchanged with ¹H at amide sites; ω_0H/2π = 1000 MHz.

Figure 5

¹³C–¹³C MAS spectrum of ILFY-labeled M2_18–60 recorded at ω_0H/2π = 900 MHz and ω_r/2π = 20 kHz using PAR mixing with τ_mix = 15 ms. A 4-fold molar excess of rimantadine drug was present in the sample and produced minimal perturbations of the chemical shifts, as expected from an S31N M2_18–60.

Figure 6

C_α–C_α region of a PDSD spectrum with τ_mix = 400 ms recorded at ω_0H/2π = 750 MHz and ω_r/2π = 14.287 kHz. A 4-fold molar excess of rimantadine drug was present in the sample and did not perturb the chemical shifts of the S31N mutant.

Figure 7

¹⁵N–¹³C aliphatic correlations from a ¹⁵N–¹³C ZF-TEDOR spectrum of a 1:1 mixture of ¹⁵N and 1,6-¹³C glucose-labeled M2_18–60 recorded with τ_mix = 14.3 ms.

Figure 8

¹³C–¹³C RFDR spectrum of 1,6-¹³C glucose-labeled M2_18–60 showing interhelical cross-peaks (red labels) inter-residue cross-peaks (green), and intraresidue cross-peaks (black). Gray labels indicate cross-peaks that can only be explained due to the presence of intertetramer contacts. The spectrum was acquired with broad-band RFDR at τ_mix = 8 ms, ω₁/2π = 100 kHz of ¹H TPPM decoupling, ω_r/ 2π = 20 kHz, and ω_0H/2π = 900 MHz.

Figure 9

Timing diagram for the 4D ¹³C²H₂¹H–¹³C²H₂¹H distance in the HCHHCH spectrum shown in Figure 10. Narrow and wide pulses represent 90 and 180° pulses, respectively. The delay Δ was optimized for transfer via the C–H J-coupling. RFDR and H₂O suppression blocks were looped to reach the correct time. Low-power TPPM or WALTZ decoupling was used during ¹H acquisition. Phases were X unless indicated, and ϕ₁ = 13, ϕ₂ = 1111 3333, ϕ₃= 0022, and ϕ_rec = 0220 2002.

Figure 10

Methyl spectra of M2_18–60 labeled with ¹³C²H₂¹H groups in the I, L, and V residues. The J-transferred 2D spectrum of the stereospecifically ¹³C²H₂¹H-labeled sample is shown in A, with assignments of isoleucine Cδ1, leucine Cδ2, and valine Cγ2 methyl groups. Notice the excellent resolution and the peak doubling observed in other ¹³C/¹⁵N spectra. B–E show selected planes from a ¹H-detected 4D using ¹H–¹H RDFR and τ_mix = 8 ms and J-coupling-based transfers for ¹³C–¹H correlation. Interhelical correlations are shown in green. Autocorrelations are indicated with asterisks. Details of the pulse sequence are shown in Figure 9.

Figure 11

Helical wheel representation of a two-fold symmetric M2 tetramer depicting the set of interhelical restraints (dashed lines) used in the final annealing, after resolution of ambiguities. The side chains of important residues N31, H37, and W41 are indicated with solid black lines.

Figure 12

Dimer of dimers structure of M2. (A) Ensemble of seven low-energy structures. The backbone and all heavy atom rmsd values are 0.7 and 1.1 Å, respectively. (B) Surface representation showing a continuous hydrophobic exterior. (C) Pore surface as calculated using the program HOLE, colored in red, green, and blue for pore widths of <1 water, 1 water, and >1 water, respectively. (D) C-terminal view of the pore with H37 in red and W41 in blue in two distinct side chain conformations. W41 adopts an “indole in” conformation for one helix and an “indole out” conformation in the other. Unstructured N- and C-terminal residues are not displayed.

Figure 13

(A) Assembly of four H37 and W41 residues within the dimer of dimers is shown. A short intermolecular distance of 3–3.5 Å is observed between H37′ and W41 in the ensemble of calculated structures calculated from the ¹³C–¹⁵N and some of the ¹H detected data. (B) This proximity was subsequently confirmed by an (H)NHH_RFDR spectrum recorded showing H37′–W41 cross peaks recorded with τ_mix = 3.33 ms of ¹H–¹H RFDR recoupling. The buildup of this peak is shown for a range of mixing times together with other assigned crosspeaks including a ¹H–¹⁵N backbone distance a calibration.

Figure 14

S31N and drug-bound structures explain drug resistance.The S31N structure in lipids (A) places N31 in the pore or in the helix–helix interface. The WT chimera structure (B) with bound drug (pdb code 2LJC) places S31 in the helix–helix interface pointing out toward the lipid membrane mimetic. Drug is shown in magenta, the drug-binding pocket (V27 and A30) is depicted as green sticks, and residue 31 (serine or asparagine) is shown as spheres.

Figure 15

A possible conduction mechanism consistent with the close proximity of H37′ and W41 found in the dimer of dimers structure (Figure 13A). The black H represents a proton that enters the channel from the n-terminal side. The blue H represents a proton transferred from H47 to W41. The red H represents the proton that can be readily released into the virus. Tautomerization of the H37 side-chain brings the protein back to the original state.