Specificity determining or “signature” positions are amino acids directly involved in an enzyme’s activity.

<p>Three such residues are shown as black side chains (left) for 5 arsenate reductase enzymes. Residues within close structural space are shown as colored fragments. The sequences of those colored fragments create the active site signature (right), a residue sequence originally defined by Cammer and colleagues [<a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005756#pcbi.1005756.ref035" target="_blank">35</a>]. Signatures can be aligned to create a profile, which allows direct comparison of residues in and near the active site. This active site profile concept is used by 3 of the approaches for functionally relevant clustering of protein superfamilies included in this Focus Feature. (The authors gratefully acknowledge Mikaela Rosen for creating these figures).</p

A Global Comparison of the Human and <em>T. brucei</em> Degradomes Gives Insights about Possible Parasite Drug Targets

<div><p>We performed a genome-level computational study of sequence and structure similarity, the latter using crystal structures and models, of the proteases of <em>Homo sapiens</em> and the human parasite <em>Trypanosoma brucei</em>. Using sequence and structure similarity networks to summarize the results, we constructed global views that show visually the relative abundance and variety of proteases in the degradome landscapes of these two species, and provide insights into evolutionary relationships between proteases. The results also indicate how broadly these sequence sets are covered by three-dimensional structures. These views facilitate cross-species comparisons and offer clues for drug design from knowledge about the sequences and structures of potential drug targets and their homologs. Two protease groups (“M32” and “C51”) that are very different in sequence from human proteases are examined in structural detail, illustrating the application of this global approach in mining new pathogen genomes for potential drug targets. Based on our analyses, a human ACE2 inhibitor was selected for experimental testing on one of these parasite proteases, TbM32, and was shown to inhibit it. These sequence and structure data, along with interactive versions of the protein similarity networks generated in this study, are available at <a href="http://babbittlab.ucsf.edu/resources.html">http://babbittlab.ucsf.edu/resources.html</a>.</p> </div

The <i>T. brucei</i> M32 protease model shows active site similarity to a human protease ACE2.

<p>The model of the <i>T. brucei</i> M32 protease (TbM32m, purple) is shown structurally aligned with crystal structure ACE2 (PDB code 1R4L, yellow). Depicted in ball-and-stick representation near the zinc ion are the metal binding residues and catalytic glutamate. ACE2 inhibitor MLN4760 is shown in green and ACE inhibitor lisinopril is in orange stick format (the position of which is from a structural alignment of ACE (1O86) with ACE2). The predicted steric clash of R273 in the ACE2 S1 pocket with lisinopril is marked with an arrow. The R273 CZ of ACE2 is predicted to be 1.5 Å from the lisinopril C9, so that a terminal nitrogen of R273 is in position to overlap with an oxygen of lisinopril. The arginine (R348) from TbM32m that is predicted to be close to the ACE2 R273 is also in ball-and-stick representation. The inset shows the overall structural similarity of the two proteins.</p

Distribution by catalytic type of peptidases predicted to be active in humans and <i>T. brucei</i>.

<p>In humans, proteases of catalytic type S (where the catalytic moiety is serine) is dominant, but metallo (type M) and cysteine (type C) peptidases are also abundant. In contrast, in <i>T. brucei,</i> serine peptidases are less abundant, and cysteine and metallo proteases are equally prominent. Other main catalytic types in each organism include the threonine (type T) and aspartatic (type A) proteases. Catalytic types were assigned by catalytic type designated in the family of the closest BLAST hits to MEROPS sequences.</p

TbM32 is inhibited by 28FII (ACE2 inhibitor) and not by lisinopril (ACE inhibitor).

<p>The chart shows results from a representative experiment with 1,10P (1,10 Phenanthroline, 100 µM), lisinopril (10 µM), and 28FII (10 µM). ** indicates significant difference from the control (DMSO vehicle) at p<0.005. The positive control 1,10P is a metal chelator that inhibits metallopeptidases.</p

Structural similarity network of human and <i>T. brucei</i> proteases labeled by clan.

<p>The same network as in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001942#pntd-0001942-g003" target="_blank">Figure 3</a> is colored here by assigned MEROPS clan (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001942#s2" target="_blank">Methods</a>). One cluster is composed of multiple clans (MC, MF, MH, and CF).</p

Structure similarity network of human and <i>T. brucei</i> proteases using crystal structures and models.

<p>Nodes represent experimentally characterized (crystal structure) or modeled structures and edges represent pairwise structural similarity above the structural similarity threshold (FAST SN score ≥4.5). Nodes for 342 human and 71 <i>T. brucei</i> are shown in the network (total of 413 nodes and 7,234 edges). The two <i>T. brucei-</i>specific families (TbM32 and C51) highlighted in the sequence similarity network shown in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001942#pntd-0001942-g001" target="_blank">Figure 1</a> are circled in red. (A) Nodes are colored by MEROPS-associated family, revealing cross-family structural relationships. Human structures are represented as circles and <i>T. brucei</i> as triangles. (B) The same structure similarity network as in panel A is painted by species and structure representation. Nodes are color-coded by species and node shape corresponds to type of structure representation for that sequence: square = crystal structure; triangle = ModBase model; diamond = ModWeb model. In contrast to <i>T. brucei,</i> there are a large number of experimentally characterized (crystal) structures for humans, but many <i>T. brucei</i> structures can be modeled.</p

Structure alignment of <i>T. brucei</i> C51 model (TbC51m) with a distant structure homolog, human Cathepsin F (CatF).

<p>The superposition shows these two proteins have some general, overall structural similarities, but also large differences near the active site. The TbC51 model is colored in light orange, and the human CatF is in light green. While the catalytic Cys-His dyads are closely superimposed (depicted in ball-and-stick), a striking difference is marked by an arrow indicating the predicted steric clash between the CatF vinyl sulfone inhibitor (red) and the helix of TbC51 that partially obstructs the active site.</p

Mechanistic and Bioinformatic Investigation of a Conserved Active Site Helix in α‑Isopropylmalate Synthase from <i>Mycobacterium tuberculosis</i>, a Member of the DRE-TIM Metallolyase Superfamily

The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the <i>C</i>-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily