Activation of procathepsin H.

<p><b>A</b>. Coomassie gel of proCTSH incubated for 3 hours in sodium acetate(NaAC) buffer at various pHs (4.5, 5.0, 5.5), MES buffer at various pHs (5.5, 6.0, 6.5), thermolysin activated CTSH (TH), human liver purified CTSH (HS), and protein ladder (LD). Arrow indicates mature CTSH product. <b>B.</b> Assessment of CTSH enzymatic activity from samples in (A) and thermolysin alone (T). Data shown is relative fluorescence units (RFUs) from excitation emission readings at 360/480nm. <b>C</b>. Coomassie gel of procathepsin H (PH), proCTSH activated with CTSL for various minutes (5, 20, 60), thermolysin activated CTSH(TH) and protein ladder (LD). Arrow indicates mature CTSH product. <b>D</b>. Assessment of R-AMC (cathepsin H substrate) cleavage from proCathepsinH (proCTSH), CTSL, thermolysin (Therm), proCTSH activated with CTSL (CTSL CTSH), and thermolysin activated CTSH (Therm CTSH). Data shown is relative fluorescence units (RFUs) from excitation emission readings at 360/480nm.</p

Assessment of cathepsin H point mutants.

<p>(A) Measurement of cathepsin H aminopeptidase activity in HEK293T cell lines overexpressing the indicated forms of cathepsin H. Activity is expressed as RFUs (relative fluorescent units) detected after cleavage of cathepsin H substrate. (B) Western blot analysis of cathepsin H and actin in HEK293T cell lines expressing the indicated forms of cathepsin H. Arrow indicates pro form and mature form of cathepsin H.</p

Crystal structure of procathepsin H.

<p>(A) Overall structure of procathepsin H. Left, the prodomain and the mature domain are colored in violet and cyan respectively. The mini-chain is colored in blue. The prodomain contains two conserved sequence motifs: ERFNIN motif (orange) and GNFD motif (green). The four disulfide bonds in the structure are highlighted in yellow.Right, the 2<i>F</i>_<i>o</i>-<i>F</i>_<i>c</i> electron density map (top) and <i>F</i>_<i>o</i>-<i>F</i>_<i>c</i> omit map (bottom) of the mini-chain are displayed as the grey mesh at a contour level of 1.2<i>σ</i> and 3<i>σ</i> respectively. (B) Superposition of procathepsin H (cyan) and procathepsin L (violet). (C) Superposition of human procathepsin H (cyan) and mature porcine cathepsin H (wheat). The mini-chain of human procathepsin H is shown in blue and that of mature porcine cathepsin H is shown in orange. (D) Hydrogen bonding interactions between the β strand from prodomain (violet) and β strand from right subdomain (cyan). Backbones of the residues involved are shown as sticks. The distances between atoms are indicated by dashes. (E) Interactions between hydrophobic residues from prodomain (violet) and the mature domain (cyan). Sidechains of the residues involved are shown as sticks. (F) Hydrogen bonding interactions between the C-terminal linker of prodomain (violet) and core enzyme (cyan). Backbones and sidechains of the residues involved are shown as sticks. The distances between atoms are indicated by dashes.</p

The stability of the mini-chain in cathepsin H and procathepsin H.

<p>The snapshots of the mini-chain and mature domain complexes before simulations (<i>left</i>) and the overlay of the mini-chain conformations during the simulations (<i>right</i>) in systems (A) 8pch_1glyc, (B) 6czk_1glyc, and (C) 6czk_2glyc. The cathepsin H (orange) and the procathepsin H (blue) systems are distinguished by their colors. The glycans present are shown in stick representation. (D) The per-residue RMSF of the mainchain atoms in the mini-chain calculated from the simulations and the B-factors from the crystal structures averaged by residue. The three systems 8pch_1glyc (filled orange circle), 6czk_1glyc (empty blue circle), and 6czk_2glyc (filled blue circle) are shown in different styles. The B-factors for 8PCH (orange) and 6CZK (blue) are shown as lines. (E) The distribution of native (left) and total (right) contact numbers computed from the simulations. Two residues are identified as contacting residues when any of the heavy atoms from the two are within 4.5 Å. The total contact number is evaluated for all residue pairs between the mini-chain and the mature domain. The native contacts are the ones from the total contacts that are also present in the native crystal structure. Results from the three systems 8pch_1glyc (solid orange line), 6czk_1glyc (dashed blue line), and 6czk_2glyc (solid blue line) are shown.</p

Crystallographic data and refinement statistics.

<p>Crystallographic data and refinement statistics.</p