Phylogenetic tests of positive selection for target proteases.

<p>−2Δ<i>L</i>, likelihood ratio test to detect positive selection; p: proportion of sites under positive selection for M8 model; ω^a: dN/dS estimate for M0; ω^b: dN/dS estimate for M8 model; ^cPositively selected sites identified by M8 model: amino acid sites found to be under positive selection with posterior probabilities greater than 90% (blank), 95% (underlined) or 99% (bold) in the BEB analysis. ^a The reference sequence is human SERPINB3. NA, not applicable because the neutural model fits better than positive selection.</p><p>** Significance with p<0.001.</p><p>* Significance with p<0.05.</p

Likelihood ratio test for branch-site model for target proteases using <i>H. sapiens</i>, <i>P. troglodytes</i> and <i>G. gorilla</i> lineage as foreground.

<p>−2Δ<i>L</i>, likelihood ratio test to detect positive selection; Foreground: <i>H. sapiens</i>, <i>P. troglodytes</i> and <i>G. gorilla</i> lineage. *Significance with p<0.05; Positively selected sites, amino acid sites found to be under positive selection with a posterior probabilities greater 90%; NA, not applicable because the neutral model fits better than positive selection.</p

X-ray structure of SERPINB3 and predicted structure of SERPINB4.

<p>The A β-sheet (shutter) is in orange, B β-sheet (breach) is in red and C β-sheet (gate) is in blue. Helices are shown in green. RCL: reactive center loop. Sites under positive selection are in black.</p

Likelihood ratio test for branch-site model for SERPINB3/B4 phylogeny.

<p>−2Δ<i>L</i>, likelihood ratio test to detect positive selection with 1 degree of freedom; Foreground 1: <i>H. Sapiens</i> B3, <i>P. Troglodytes</i> B3 and <i>G. Gorilla</i> B3 lineages; Foreground 2: <i>H. Sapiens</i> B4, <i>P. Troglodytes</i> B4 and <i>G. Gorilla</i> B4 lineages. Amino acid sites found to be under positive selection with posterior probabilities greater than 80% (blank) are displayed; NA, not applicable because the neutral model fits better than positive selection.</p><p>** Significance with p<0.01.</p

Adaptive Evolution and Divergence of <i>SERPINB3</i>: A Young Duplicate in Great Apes

<div><p>A series of duplication events led to an expansion of clade B Serine Protease Inhibitors (<i>SERPIN</i>), currently displaying a large repertoire of functions in vertebrates. Accordingly, the recent duplicates <i>SERPINB3</i> and <i>B4</i> located in human 18q21.3 <i>SERPIN</i> cluster control the activity of different cysteine and serine proteases, respectively. Here, we aim to assess SERPINB3 and B4 coevolution with their target proteases in order to understand the evolutionary forces shaping the accelerated divergence of these duplicates. Phylogenetic analysis of primate sequences placed the duplication event in a Hominoidae ancestor (∼30 Mya) and the emergence of <i>SERPINB3</i> in Homininae (∼9 Mya). We detected evidence of strong positive selection throughout <i>SERPINB4</i>/<i>B3</i> primate tree and target proteases, cathepsin L2 (<i>CTSL2</i>) and G (<i>CTSG</i>) and chymase (<i>CMA1</i>). Specifically, in the Homininae clade a perfect match was observed between the adaptive evolution of <i>SERPINB3</i> and cathepsin S (<i>CTSS</i>) and most of sites under positive selection were located at the inhibitor/protease interface. Altogether our results seem to favour a coevolution hypothesis for <i>SERPINB3</i>, <i>CTSS</i> and <i>CTSL2</i> and for <i>SERPINB4</i> and <i>CTSG</i> and <i>CMA1</i>. A scenario of an accelerated evolution driven by host-pathogen interactions is also possible since SERPINB3/B4 are potent inhibitors of exogenous proteases, released by infectious agents. Finally, similar patterns of expression and the sharing of many regulatory motifs suggest neofunctionalization as the best fitted model of the functional divergence of <i>SERPINB3</i> and <i>B4</i> duplicates.</p></div

The best docking for SERPINB3 (green)/CTSS (blue) and SERPINB4 (green)/CTSG (blue) complex models generated using HADDOCK software.

<p>Amino acids under positive selection at the SERPIN/protease interface are in black. Amino acids at the inhibitor scissile bond and forming the proteases catalytic triad are depicted in red. Arrows point the location of β-sheet A (SA), β-sheet B (SB) and β-sheet C (SC). Binding regions are enlarged for a more detailed view (left panel).</p

Inhibitor protein complexes tested by docking analysis.

<p>i-RMSD: interfacial root mean square deviation; l-RMSD: ligand root mean square deviation; HADDOCK score is weighted sum of van der Waals, electrostatic, desolvation and restrained violation energies together with buried surface area.</p

Origin of <i>SERPINB3</i> and <i>SERPINB4</i> duplicates.

<p>A) The organization of <i>SERPINB3</i> and <i>SERPINB4</i> loci in human and eight non-human primates. Relative position to telomere (Tel) and centromere (Cen) is shown. Solid boxes represent functional genes; open boxes represent pseudogenes. B) Phylogenetic tree of <i>SERPINB3</i> and <i>SERPINB4</i> genes with the bootstrap percentages shown at interior nodes and the alignment of RCL regions (P17-P4′). The canonical scissile bond is marked by an arrow and a standard P1 and P1′ nomenclature is used to number amino acid positions N- and C-terminal outward from the scissile bond. AncB3/B4: ancestral SERPINB3/B4 gene.</p

Expression patterns of SERPINB3 and SERPINB4 in human tissues.

<p><i>GAPDH</i> amplification was used as a control. NC: negative control.</p

Maximum likelihood estimates of positive selection for SERPINB3/B4 phylogeny.

<p>Likelihood ratio tests (−2Δ<i>l</i>) comparing a null and positive selection models (M1a vs M2a, M7 vs M8); N, number of primate species with sequences in alignment; p: proportion of sites under positive selection in M8 model; ω: estimate the dN/dS of the sites under selection in M8 model; ^a Amino acid sites found to be under positive selection with posterior probabilities greater than 90% (blank), 95% (underlined) or 99% (bold) in the BEB analysis. The reference sequence is human SERPINB3.</p><p>** Significance with p<0.001.</p