Cip1 pocket that binds ethylene glycol.

<p>With Arg100 (lime green) forming one of the walls, Thr85, Glu194, His83 and Tyr196 together create the rest of a small pocket on one side of the plausible active site cleft, in which an ethylene glycol (dark green) is found in the structure of Cip1. To facilitate comparison of figures, Gln104 is also shown (lime green). Electron density is contoured at a level of 1.0 sigma (0.4 electrons/ų).</p

The calcium binding site in Cip1 compared to glucuronan lyase from <i>H.</i><i>jecorina</i>.

<p>The calcium binding site found in the Cip1 structure. Cip1 structure (green) superposed to the glucuronan lyase structure from <i>H. jecorina</i> (red). Asp206 is shown in bright colours since it is sequentially and structurally conserved and it coordinates the calcium ion with the two side chain oxygen atoms (also see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone-0070562-g008" target="_blank">Figure 8</a>). All coordination distances are between 2.3 Å and 2.6 Å.</p

The Crystal Structure of the Core Domain of a Cellulose Induced Protein (Cip1) from <i>Hypocrea jecorina</i>, at 1.5 Å Resolution

<div><p>In an effort to characterise the whole transcriptome of the fungus <i>Hypocrea jecorina</i>, cDNA clones of this fungus were identified that encode for previously unknown proteins that are likely to function in biomass degradation. One of these newly identified proteins, found to be co-regulated with the major <i>H. jecorina</i> cellulases, is a protein that was denoted Cellulose induced protein 1 (Cip1). This protein consists of a glycoside hydrolase family 1 carbohydrate binding module connected <i>via</i> a linker region to a domain with yet unknown function. After cloning and expression of Cip1 in <i>H. jecorina</i>, the protein was purified and biochemically characterised with the aim of determining a potential enzymatic activity for the novel protein. No hydrolytic activity against any of the tested plant cell wall components was found. The proteolytic core domain of Cip1 was then crystallised, and the three-dimensional structure of this was determined to 1.5 Å resolution utilising sulphur single-wavelength anomalous dispersion phasing (sulphor-SAD). A calcium ion binding site was identified in a sequence conserved region of Cip1 and is also seen in other proteins with the same general fold as Cip1, such as many carbohydrate binding modules. The presence of this ion was found to have a structural role. The Cip1 structure was analysed and a structural homology search was performed to identify structurally related proteins. The two published structures with highest overall structural similarity to Cip1 found were two poly-lyases: CsGL, a glucuronan lyase from <i>H. jecorina</i> and vAL-1, an alginate lyase from the <i>Chlorella</i> virus. This indicates that Cip1 may be a lyase. However, initial trials did not detect significant lyase activity for Cip1. Cip1 is the first structure to be solved of the 23 currently known Cip1 sequential homologs (with a sequence identity cut-off of 25%), including both bacterial and fungal members.</p></div

Topology diagram of Cip1.

<p>Secondary structure of <i>Hypocrea jecorina</i> Cip1 coloured in rainbow from N-terminal blue to C-terminal red. The concave active site cleft β-sheet is on the right in the topology diagram (β-sheet B). The “grip” motif is on the left, in part consisting of the outer convex β-sheet “palm” (β-sheet A) and the “bent fingers” formed by the loop of residues 32–41. The calcium ion is depicted in grey and coordinates residues from both the N-terminal and C-terminal as well as from the loop in the grip motif, thereby stabilizing the structure in that area.</p

Thermal unfolding of Cip1.

<p>Panel <b>A</b> shows two different curves, one showing pH dependence of the thermal unfolding midpoints (Tm; •) and the other showing pH dependence of the reversibility of the amplitude of unfolding for Cip1 (o). The differential scanning calorimetry profiles were collected over pH range of 3.2-to-8.8. The data was collected from 30–90°C at a scan rate of 200°C/hr using the VP-Cap DSC (MicroCal, Inc. Northampton, MA). The reversibility of the unfolding amplitudes was calculated using Peakfit v.4.12 (Seasolve Software, Inc, MA). The solid lines are to guide the eye. Panel <b>B</b> shows the thermal unfolding profiles for Cip1 at pH 6.8 in the absence (A) and presence (B) of 5 mM ethylene-diamine-tetra-acetate (EDTA). Rescans of the thermally unfolded samples in the absence (C) and presence (D) of EDTA are also shown. All scans were performed at 200°C/hr over a temperature range of 30–90°C using Auto-Cap DSC (MicroCal, Northampton, MA).</p

Overall view of Cip1.

<p>Overall view of <i>Hypocrea jecorina</i> Cip1 showing the structure in A) front view and B) side view. The β-strands that make up the bottom of the cleft (β-sheet B) are coloured in red, forming a β-sandwich together with β-sheet A (green). A red circle surrounds the “grip” motif where a calcium ion is also found (blue).</p

Sequence alignment of Cip1 homologs.

<p>Sequence alignment of <i>H. jecorina</i> Cip1 amino acid sequence with all publically available protein sequences with a BLAST identity percentage of at least 25%. Sequences 1–10 are fungal sequences and sequences 11–24 are from bacteria. The residues marked in green are located in the “grip” region (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone-0070562-g008" target="_blank">fig. 8</a>), the residues marked in bright orange are plausible active site residues in the cleft of the structure, the light orange residues are located together on one side of the cleft interacting with an ethylene glycol molecule in the Cip1 structure and the residues marked in yellow interact with a calcium ion in the “grip” region of Cip1. The secondary structure is marked with boxes and each element coloured according to the rainbow colouring in the related topology diagram (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone-0070562-g003" target="_blank">fig. 3</a>). The shown aligned sequences (EMBL Genbank access numbers indicated in parentheses) are: seq. 1, <i>Hypocrea jecorina</i> Cip1 (AAP57751); seq. 2, <i>Pyrenophora teres f teres</i> 0–1 (EFQ89497); seq. 3, <i>Pyrenophora tritici repentis</i> (XP_001937765); seq. 4, <i>Chaetomium globosum</i> (XP_001228455); seq. 5, <i>Chaetomium globosum</i> (XP_001222955); seq. 6, <i>Phaeosphaeria nodorum</i> SN15 (XP_001790983); seq. 7, <i>Podospora anserina</i> S mat+ (XP_001906367); seq. 8, <i>Magnaporthe oryzae</i> 70-15 (XP_365869); seq. 9, <i>Nectria haematococca</i> mpIV (XP_003039679); seq. 10, <i>Gibberella zeae</i> PH-1 (XP_386642); seq. 11, <i>Haliangium ochraceum</i> DSM 14365 (YP_003266142); seq. 12, <i>Herpetosiphon aurantiacus</i> ATCC 23779 (YP_001545140); seq. 13, <i>Catenulispora acidiphila</i> DSM 44928 (YP_003114993); seq. 14, <i>Streptomyces coelicolor</i> A3(2) (NP_629910); seq. 15, <i>Streptomyces lividans</i> TK24 (ZP_05523220); seq. 16, <i>Streptomyces sp</i>. ACTE (ZP_06272077); seq. 17, <i>Streptomyces sviceus</i> ATCC 29083 (ZP_06915571); seq. 18, <i>Streptomyces sp</i>. e14 (ZP_06711846); seq.19, <i>Actinosynnemma mirum</i> DSM 43827 (YP_003101274); seq. 20, <i>Amycolatopsis mediterranei</i> U32 (YP_003767350); seq. 21, <i>Streptomyces violaceusniger</i> Tu 4113 (ZP_07602526); seq. 22, <i>Cellulomonas flavigena</i> DSM 20109 (YP_003638201); seq. 23, <i>Micromonospora aurantiaca</i> ATCC 27029 (YP_003835070); seq. 24, <i>Micromonospora sp.</i> L5 (YP_004081730).</p

Diffraction data, processing, phasing and structure refinement statistics.

a<p>Beamlines at the European Synchrotron Radiation Facility (ESRF), Grenoble, France.</p>b<p>Numbers in parentheses are for the highest resolution bins.</p><p>The table values were calculated with O <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone.0070562-Jones1" target="_blank">[41]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone.0070562-Jones2" target="_blank">[46]</a>, Refmac5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone.0070562-Murshudov1" target="_blank">[37]</a>, CNS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone.0070562-Brnger2" target="_blank">[47]</a>, MOLEMAN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone.0070562-Kleywegt3" target="_blank">[48]</a>, and LSQMAN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone.0070562-Kleywegt4" target="_blank">[49]</a>. Calculated using the strict boundary Ramachandran definition given by Kleywegt and Jones <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070562#pone.0070562-Kleywegt1" target="_blank">[9]</a>.</p

Comparison of Cip1 to alginate lyase from <i>Chlorella</i> virus at pH 7 and pH 10.

<p>Superposition of Cip1 from <i>H. jecorina</i> (green) to the alginate lyase from <i>Chlorella</i> virus (blue) and the interactions with bound D-glucuronic acid (violet) at A) pH 7 and B) pH 10. The residues are numbered according to the Cip1 structure. Plausible catalytic residues are brightly coloured in the figure. Water molecules are depicted in red and belong to the structure of Cip1. Panel <b>A</b> displays the alginate lyase structure at pH 7, the D-glucuronic acid interacts with the glutamine at the top of the active cleft. The corresponding glutamine in Cip1 (Gln104) instead forms a hydrogen bond to a water molecule, which is also bound by Asp116, a residue that has dual conformations in Cip1. Panel <b>B</b> displays the alginate lyase structure at pH 10, the D-glucuronic acid interacts with Arg100 at the lower end of the cleft. Both Asp116 and His98 in Cip1 show dual conformations pointing toward this position which may be an indication that the region is dynamic and that these residues are somehow involved in substrate binding. Asp116 and His98 do not have any equivalents in the lyase structure.</p