Communication breakdown : dissecting the COM interfaces between the subunits of nonribosomal peptide synthetases

Nonribosomal peptides are a structurally diverse and bioactive class of natural products constructed by multidomain enzymatic assembly lines known as nonribosomal peptide synthetases (NRPSs). While the core catalytic domains and even entire protein subunits of NRPSs have been structurally elucidated, little biophysical work has been reported on the docking domains that promote interactions—and thus transfer of biosynthetic intermediates—between subunits. In the present study, we closely examine the COM domains that mediate COMmunication between donor epimerization (E) and acceptor condensation (C) domains found at the termini of NRPS subunits. Through a combination of X-ray crystallography, circular dichroism spectroscopy, solution- and solid-state NMR spectroscopy, and molecular dynamics (MD) simulations, we provide direct evidence for an intrinsically disordered donor COM region that folds into a dynamic helical motif upon binding to a suitable acceptor. Furthermore, our NMR titration and carbene footprinting experiments illuminate the residues involved at the COM interaction interface, and our MD simulations demonstrate folding consistent with experimental data. Although our results lend credence to the previously proposed helix-hand mode of interaction, they also underscore the importance of viewing COM interfaces as dynamic ensembles rather than single rigid structures and suggest that engineering experiments should account for the interactions which transiently guide folding in addition to those which stabilize the final complex. Through activity assays and affinity measurements, we further substantiate the role of the donor COM region in binding the acceptor C domain and implicate this short motif as readily transposable for noncognate domain crosstalk. Finally, our bioinformatics analyses show that COM domains are widespread in natural product pathways and function at interfaces beyond the canonical type described above, setting a high priority for thorough characterization of these docking domains. Our findings lay the groundwork for future attempts to rationally engineer NRPS domain–domain interactions with the ultimate goal of generating bioactive molecules

Activity of bovine DNaseI and its two fusion variants at differentionic strengths.

<p>Two series of NaCl gradients were used: the first one corresponds to a range from 0 to 1 M with increments of 0.1 M (left electrophoregrams), the second one corresponds to a range from 0 to 4 M with increments of 0.4 M (right electrophoregrams). Activity of analysed proteins was evaluated by digestion of linearised pUC19 plasmid in the presence of various concentrations of NaCl. The data on the fusion with the (HhH)₂ domain of DNase from <i>Thioalkalivibrio sp. K90mix</i> is given at the C and D panels. The data on the fusion with homologous domain of ComEA protein from <i>Bacillus subtilis</i> is given at the E, F panels. The data on bovine DNaseI are given at the A, B panels. The electrophoregrams on DNaseI have been already published together with data on DNase from <i>Thioalkalivibrio sp. K90mix</i>[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150404#pone.0150404.ref006" target="_blank">6</a>].</p

Superposition of structural models of two (HhH)₂ domains from <i>Thioalkalivibrio sp. K90mix</i> (blue colour) and <i>Bacillus subtilis</i> (green colour) and corresponding sequence alignment.

<p>DNA phosphate contacting positive residues are indicated by red colour in the sequence alignment and by stick representations in the structural alignment. Yellow colour indicates part of the domain from <i>Bacillus subtilis</i>, which has no corresponding residues in the domain from <i>Thioalkalivibrio sp. K90mix</i>. Approximate position of DNA is indicated by transparent sticks based on structure PDBID: 3E0D after superimposition with the domains.</p

Yield of DNaseI fusions and general characterisation of used protein samples.

<p>DNaseDT denotes the fusion with the (HhH)₂ domain of DNase from <i>Thioalkalivibrio sp. K90mix</i>. DNaseBS—the fusion with homologous domain of ComEA protein from <i>Bacillus subtilis</i>.</p

Radioactive substrate digestion half-life of wild type bovine DNaseI and its fusion variants.

<p>DNaseDT denotes the fusion with the (HhH)₂ domain of DNase from <i>Thioalkalivibriosp. K90mix</i>. DNaseBS—the fusion with homologous domain of ComEA protein from <i>Bacillus subtilis</i>.</p

Changes in polar electrostatic solvation energy upon complex formation at different NaCl concentrations.

<p>Two domains were modeled: one from an extremely salt tolerant bacterium <i>Thioalkalivibrio sp. K90mix</i> (DT), the other one from <i>Bacillus subtilis</i>(BS).</p

Electrostatic surface potential of DNA-binding surface and changes in local ion concentration upon binding to DNA.

<p>Electrostatic potential of DNA-binding surface of the domain from <i>Bacillus subtilis</i> is shown on the upper-left (A) and corresponding surface of the domain from <i>Thioalkalivibrio sp. K90mix</i> is given on the upper-right (B). The range from −1.5 kT/e in red to +1.5 kT/e in blue was chosen for surface colouring. The surface is semi transparent and stick representations of the DNA phosphates contacting residues are visible: lysines—in case of <i>Bacillus subtilis</i> domain and arginines in case of <i>Thioalkalivibrio sp. K90 mix</i> domain. The changes in local ion concentrations upon formation of a complex with DNA by the domain from <i>Bacillus subtilis</i> are depicted in the lower-left (C), the corresponding changes in the case of the domain from <i>Thioalkalivibrio sp. K90mix</i> are depicted in the lower-right (D). The DNA phosphates interacting residues are depicted by white sticks. The isocountour surfaces indicate the changes, which occur in the presence of 0.4 M NaCl. Four isocountour surfaces are visualized simultaneously. The deep blue resembles changes in local ion concentration equivalent to −3 M, the lighter blue resembles changes equivalent to −2 M. Similarly deep red indicates changes equivalent to +3 M and the lighter red indicates changes equivalent to +2 M. Isosurfaces equivalent to +2/-2 M overlaps corresponding isosurfaces which resemble changes equivalent to +3/-3 M.</p

Efficiency of DNA removal by DNaseI and its fusion variants during RNA purification procedure.

<p>DNaseDT denotes the fusion with the (HhH)₂ domain of DNase from an extremely salt tolerant bacterium <i>Thioalkalivibrio sp. K90mix</i>, DNaseBS denotes the fusion with homologous domain of ComEA protein from <i>Bacillus subtilis</i>. The upper picture represents quantitative evaluation of undigested DNA remaining in eluates (RT-qPCR without added reverse transcriptase). The lower picture represents RT-qPCR results obtained using the same eluates. In the both pictures labels on the horizontal axis indicate the used nuclease, amount of it and the dilution ratio of the eluates before reverse transcription step and quantitation of DNA. A modified protocol of GeneJET™ Whole Blood RNA Purification Mini Kit (Thermo Fisher Scientific, #K0761) was followed and RNA purification columns supplied by the manufacturer were used. During the experiment we have purified total blood RNA. Three arbitrary blood samples were analysed. DNA digestion was performed directly on a filter of a RNA purification column, were respective DNase enzyme was loaded.</p

Changes in ion density on the DNA surface upon complex formation.

<p>Upper part resembles performed analysis in case of the domain from <i>Bacillus subtilis</i> (A), the lower part—in case of the domain from <i>Thioalkalivibrio sp. K90mix</i> (B). The white wireframes denotes density of positive charges around the DNA molecule and resembles isosurface at +3 <i>Me</i>_<i>c</i> charge. The yellow isosurface indicates the changes in the ion density upon domain—DNA complex formation. This isosurface indicates area, where local ion concentration decreases by 3 M.</p

Hydrogen bonding network between (HhH)₂ domains and DNA.

<p>Two domains were analysed: one from an extremely salt tolerant bacterium <i>Thioalkalivibrio sp. K90mix</i>(DT), the other one from <i>Bacillus subtilis</i>(BS).</p