Promoter binding, opening, GTP binding and RNA turnover.

<p>(A) TAMRA fluorophore labeled promoter DNA (20 nM) was titrated with increasing P266L T7 RNAP (0 to 100 nM) and increase in fluorescence anisotropy was fit to the quadratic equation with <i>K</i>_d of 3.6±1.1 nM. Similar to the WT T7 RNAP, the P266L mutant did not cause significant changes in the TAMRA fluorescence intensity (<10%) upon binding. Effect of the intensity changes on the fitting was insignificant and corrected the same way as reported previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091859#pone.0091859-Tang4" target="_blank">[23]</a>. Shown here are averaged values with standard deviations (error bars) from multiple independent measurements, from which <i>K</i>_d was fitted. (B) The increase in 2-AP fluorescence at −4 in the template strand upon addition of WT and P266L indicate slightly lower promoter opening with P266L. The errors are standard deviation from 10–15 measurements. (C) Initiating GTP binding was monitored from fluorescence increase in the 2-AP (−4 position) labeled promoter DNA bound to T7 RNAP titrated with increasing 3′dGTP. The P266L binds to the initiating NTPs (3′dGTP) ∼2 times weaker than WT T7 RNAP (175 μM versus 405 μM) and Hill coefficients are 1.3±0.02 and 1.7±0.03 for WT and P266L, respectively. The errors represent the fitting uncertainty. (D) A complex of WT or P266L T7 RNAP (2 µM) and promoter DNA (1 µM) was mixed with limited NTPs or NTP and 3′-deoxy NTP mixture for 2 min at 25°C to allow RNA synthesis of the indicated lengths. The amount of RNA shown in µM is representative of the number of turnovers at each walked position for WT (black) and P266L (grey) T7 RNAP.</p

Upstream bubble collapse and transition into elongation are delayed in the P266L mutant.

<p>(A) Real time 2AP fluorescence monitors upstream bubble collapse. Representative time courses of 2-AP fluorescence changes from position −4NT in individual walking experiments (+8, +9, +12, and +15) were observed in the stopped-flow setup. The initial increase indicates rapid opening of the promoter and the decrease indicates bubble collapse. (B) Single molecule FRET histograms measure the rate of promoter unbending by WT and P266L T7 RNAP at +9. The number of DNA molecules that were analyzed to draw the smFRET histograms are as follows: P266L: 3110 at 1 min; 4164 at 5 min; 4883 at 33 min. WT: 3444 at 1 min; 3432 at 5 min; 5855 at 34 min. The <i>x</i>-axis shows corrected FRET (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091859#pone.0091859.e002" target="_blank">equation 2</a>) and the <i>y-</i>axis represents the frequency of transcription complexes with the respective FRET values. Low FRET is observed in the elongation complex (EC) and high FRET is observed in the initiation complexes (IC). An increase in the low FRET population is observed over time after stalling at +9 using GTP+ATP+CTP+3′dUTP. Concentration of T7 RNAP-DNA, GTP, and NTPs was 10 nM, 1 mM, and 500 µM, respectively. (C) The fraction of EC versus time was fit to a single exponential function. The error bars in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091859#pone-0091859-g005" target="_blank">Fig 5C</a> is the standard error from fitting smFRET histogram to a single exponential function. The WT (red circles) transitions to EC faster than P266L (black circles) at position +9 (τ = 4.5±1 min for P266L and 1.7±0.3 min for WT). The cartoon shows the layout of the smFRET experiments.</p

The P266L mutation modifies both rotation and DNA scrunching changes during initiation.

<p>(A) Cartoon illustration of FRET experiments to measure promoter rotation. The polymerase is in gray, the non-template strand in red and the template in green. Fluorescent donor TAMRA (red sphere) was introduced at position −22 in the non-template strand and acceptor Alexa 647 (blue square) at designated downstream positions on the template strand. Transcription complexes were walked to position <i>N</i> (+4 to +13) and FRET efficiency between donor (D) at −22 and acceptor (A) at <i>N</i>+<i>5</i> was measured to obtain the D-A distances (R_DA, discontinuous line). (B and C) Average FRET efficiency and changes in D-A spatial distances are shown for P266L and WT T7 RNAP (D) Cartoon illustration of FRET experiments to measure DNA scrunching. The donor Cy3 (red sphere) was labeled on position −4 and acceptor Cy5 (blue square) labeled on downstream <i>N</i>+<i>5</i> positions as above. (E and F) Average FRET efficiency and changes in averaged D-A spatial distances between Cy3 at the upstream edge (−4) and Cy5 at the downstream <i>N</i>+<i>5</i> positions with the P266L and WT T7 RNAP complexes. The error bars of FRET efficiency represent the standard deviations from multiple independent measurements (N≥3).</p

C-linker region of T7 RNAP.

<p>(A) The C-linker region (residues 251 to 296 in cartoon format) adopts different conformations in the initiation state (yellow, 1QLN, IC3), the elongation state (Pink, 1MSW, EC), and with 7 nt transcript bound (blue, 3E2E, IC7). The P266 and L266 residue is shown in stick format. The amino acids from 255 to 263 are disordered in the P266L structure (3E2E) and shown as dashed line. The direction of rotation of the linker near the hinge region is marked with arrows. The C-terminal domains (residues 300–883) of the three structures were aligned using Pymol (Molecular graphics systems). (B) Conservation of proline residue in the linker region between N-terminal domain and C-terminal domain at positions 266 and 270 in single-subunit RNAPs of phage, bacterium, and eukaryotic mitochondria. The N-terminal 1–300 amino acid sequence of T7 RNAP was used as a query in a BLAST amino acid search of the NCBI database for sequence alignment.</p

Distinct transcription initiation pathways of WT and P266L T7 RNAP.

<p>(A) The transcription initiation pathways of WT T7 RNAP (top) and P266L T7 RNAP (bottom) are shown in cartoon format to illustrate the distinct intermediate conformations. The N-terminal domain is shown in blue, C terminal domain in red, DNA in black and RNA in green. Movement of the N terminal domain is marked by the arrow. Both WT and P266L T7 RNAP bind, bend, and open the promoter DNA from −4 to +2 to the same extent. The rigid C-linker of WT favors progressive rotation of the upstream end of the promoter to accommodate the growing hybrid from +4 to +6 positions, which pushes against the N-terminal domain driving the rotation of the promoter. The pushback from the N-terminal domain destabilizes the RNA:DNA hybrid and leads to abortive synthesis in WT. The flexible C-linker of P266L mutant (bottom panel) accommodates RNA extension up to 6 nt without significant promoter/N-terminal domain rotation. The reduced DNA scrunching together with the absence of promoter rotation in the 4–6 nt RNA range in P266L suggests that the growing hybrid is accommodated by an alternative pathway. After 6 nt RNA synthesis, promoter rotation and scrunching resumes in P266L. The weakened promoter interactions in WT after 9 nt RNA synthesis allow release of the N-terminal domain and transition into elongation. Persistent promoter interactions delay the transition in P266L. (B) Template strand scrunching in P266L RNAP with 7 bp RNA:DNA. Template strand from the IC3 (PDB: 1QLN) and IC7 (PDB: 3E2E) crystal structures showing decrease in the distance between C+1 and T-3 in the IC7 structure (brown) compared to the IC3 structure (blue).</p

DataSheet_1_Zilucoplan, a macrocyclic peptide inhibitor of human complement component 5, uses a dual mode of action to prevent terminal complement pathway activation.docx

IntroductionThe complement system is a key component of the innate immune system, and its aberrant activation underlies the pathophysiology of various diseases. Zilucoplan is a macrocyclic peptide that binds and inhibits the cleavage/activation of human complement component 5 (C5). We present in vitro and ex vivo data on the mechanism of action of zilucoplan for the inhibition of C5 activation, including two clinically relevant C5 polymorphisms at R885.MethodsThe interaction of zilucoplan with C5, including for clinical C5 R885 variants, was investigated using surface plasmon resonance (SPR), hemolysis assays, and ELISA. The interference of C5b6 formation by zilucoplan was investigated by native gel analysis and hemolysis assay. The permeability of zilucoplan in a reconstituted basement membrane was assessed by the partition of zilucoplan on Matrigel-coated transwell chambers.ResultsZilucoplan specifically bound human complement C5 with high affinity, competitively inhibited the binding of C5 to C3b, and blocked C5 cleavage by C5 convertases and the assembly of the cytolytic membrane attack complex (MAC, or C5b9). Zilucoplan fully prevented the in vitro activation of C5 clinical variants at R885 that have been previously reported to respond poorly to eculizumab treatment. Zilucoplan was further demonstrated to interfere with the formation of C5b6 and inhibit red blood cell (RBC) hemolysis induced by plasmin-mediated non-canonical C5 activation. Zilucoplan demonstrated greater permeability than a monoclonal C5 antibody in a reconstituted basement membrane model, providing a rationale for the rapid onset of action of zilucoplan observed in clinical studies.ConclusionOur findings demonstrate that zilucoplan uses a dual mode of action to potently inhibit the activation of C5 and terminal complement pathway including wild-type and clinical R885 variants that do not respond to eculizumab treatment. These data may be relevant to the clinically demonstrated benefits of zilucoplan.</p