Integron Gene Cassettes: A Repository of Novel Protein Folds with Distinct Interaction Sites

Mobile gene cassettes captured within integron arrays encompass a vast and diverse pool of genetic novelty. In most cases, functional annotation of gene cassettes directly recovered by cassette-PCR is obscured by their characteristically high sequence novelty. This inhibits identification of those specific functions or biological features that might constitute preferential factors for lateral gene transfer via the integron system. A structural genomics approach incorporating x-ray crystallography has been utilised on a selection of cassettes to investigate evolutionary relationships hidden at the sequence level. Gene cassettes were accessed from marine sediments (pristine and contaminated sites), as well as a range of Vibrio spp. We present six crystal structures, a remarkably high proportion of our survey of soluble proteins, which were found to possess novel folds. These entirely new structures are diverse, encompassing all-α, α+β and α/β fold classes, and many contain clear binding pocket features for small molecule substrates. The new structures emphasise the large repertoire of protein families encoded within the integron cassette metagenome and which remain to be characterised. Oligomeric association is a notable recurring property common to these new integron-derived proteins. In some cases, the protein-protein contact sites utilised in homomeric assembly could instead form suitable contact points for heterogeneous regulator/activator proteins or domains. Such functional features are ideal for a flexible molecular componentry needed to ensure responsive and adaptive bacterial functions.13 page(s

Structure to Function: Cassette-encoded proteins from deep sea hydrothermal vent bacteria

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The Vpc_cass2 dimer (PDB 3JRT).

<p><b>A.</b> Ribbon of monomeric unit with colour spectrum (blue to red) across six helical components (α1, α2, α3, α′, α″, α4), named as indicated. A loop of weak density connecting helices 2 and 3 is represented by dotted line. <b>B.</b> Contrasting shapes of dimers of <b>Vpc_cass2</b> (left panel) and structural relative HI0074 from <i>Haemophilus influenza </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052934#pone.0052934-Lehmann1" target="_blank">[55]</a> (right panel). <b>C.</b> View (left panel) across <b>Vpc_cass2</b> dimer interface shown as electrostatic surface (in blue to red from +5 to −5 kbT/e <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052934#pone.0052934-Baker1" target="_blank">[57]</a>), highlighting the basic cluster unique to this protein. Right panel provides segmental view of dimer ribbon (green) and side chains of putative active site residues of <b>Vpc_cass2</b> straddling the dimer interface. Residues conserved in <b>Vpc_cass2</b> and its sequence homologs from <i>Shewanella</i> and <i>Moritella sp</i> are coloured orange.</p

Hfx_cass5 contains domain-swapped dimers (PDB 3IF4).

<p><b>A.</b> Ribbon depiction of two domain-swapped chains, each coloured from N-terminus (blue) to C-terminus (red). <b>B.</b> Tetrameric organisation depicted in ribbon form (left panel), showing engagement between two dimers (green and blue), each comprising two chains. Side chain stacking of key contacting residues (Tyr28, Tyr30, Arg31 and Glu35) is depicted. Corresponding view of electrostatic surface is also shown (right), with deep cleft centrally positioned. <b>C.</b> Rotated view of electrostatic surface, positioned to emphasise narrow dimensions of this basic slot along one tetramer surface.</p

Crystallographic data collection and refinement statistics for structure determination.

a<p>Chains per asymmetric unit (a.s.u).</p>b<p>∑∑<i>_i</i>|I<i>_hi −</i> I<i>_hi</i>|/∑∑<i>_i</i>I<i>_h</i>, where I<i>_h</i> is the mean intensity of reflection <i>h.</i></p>c<p>From MolProbity <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052934#pone.0052934-Davis1" target="_blank">[36]</a>.</p

The distinctive flattened shape of trimeric Hfx_cass1

<p><b>(PDB 3FUY). </b><b>A.</b> Dimensions highlight distinctive flattened form. Topology map indicates (dashed line) subfold segment found in zinc transporter domain CzrB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052934#pone.0052934-Cherezov1" target="_blank">[50]</a>, to which <b>Hfx_cass1</b> is not functionally related. <b>B.</b> Electrostatic surface potential of the trimer surface highlights polar cavities (arrowed) and exposed acidic clusters on external loops. <b>C.</b> Residues from β-strands 3 and 4 form a polar crevice (blue) surrounded by surface loops containing charged residues (red). Solvent molecules trapped within the crevice are shown (spheres).</p

Structural elements and topology of novel gene cassette proteins.

<p>Sequences of gene cassette ORFs aligned with secondary structural features observed within crystal structures (arrows, β-strands; blocks, α-helices; dashed lines, undefined flexible regions). Sequences do not include additional affinity tags used for recombinant production. Schematic diagrams show structures of monomer forms for proteins <b>A. Hfx_cass2, B. Vpc_cass2, C. Hfx_cass1, D. Hfx_cass5, E. Vch_cass3</b> and <b>F. Vch_cass14</b>.</p

Vch_cass14 dimer with sequestered ligand (PDB 3IMO).

<p><b>A.</b> Ribbon depiction of dimer coloured in spectrum from N-terminus (blue) to C-terminus (red) for each chain. <b>B.</b> Pronounced basic feature (in blue) dominates electrostatic surface of <b>Vch_cass14</b> (left); corresponding orientation of protein chain (right) outlines location of contributing Arg and Lys residues. <b>C.</b> Surface with ribbon representation (left) of the <b>Vch_cass14</b> binding pocket. Regions with ≥80% sequence conservation across the small family of homologs are coloured pink. Side chains contributing to the hydrophobic pocket are depicted. The 1.8 Å 2<i>F_o-F_c</i> map (contoured at 1σ level, mid-panel) shows side chains that line the extended cavity, as well as an area of electron density attributed to a sequestered small ligand. Residues lining this hydrophobic binding pocket are detailed (right panel). The single water molecule (red sphere) and unidentifiable ligand (grey) are also shown. The hydrogen-bonding network engaging Arg21, Tyr14, acetate and water is dashed in red.</p

Helical packing in the Hfx_cass2 dimer (PDB 3FXH).

<p><b>A</b>. Ribbon depiction with colour spectrum from N-terminus (blue) to C-terminus (red) for each chain. <b>B.</b> Dimer interface engages hydrophobic residues from Chain A (tan) and Chain B (green). Acidic groups located either side of a small hydrophobic pocket on the external face are indicated (red). <b>C.</b> Electrostatic surface potential of the dimer surface. Key acidic features (Glu47, Asp60 and helix 2 side chains) are labelled.</p