Synthesis of Aspartame by Thermolysin: An X‑ray Structural Study

Protease mediated peptide synthesis (PMPS) was first described in the 1930s but remains underexploited today. In most PMPS, the reaction equilibrium is shifted toward synthesis by the aqueous insolubility of product generated. Substrates and proteases are selected by trial and error, yields are modest, and reaction times are slow. Once implemented, however, PMPS reactions can be simple, environmentally benign, and readily scalable to a commercial level. We examined the PMPS of a precursor of the artificial sweetener aspartame, a multiton peptide synthesis catalyzed by the enzyme thermolysin. X-ray structures of thermolysin in complex with aspartame substrates separately, and after PMPS in a crystal, rationalize the reaction’s substrate preferences and reveal an unexpected form of substrate inhibition that explains its sluggishness. Structure guided optimization of this and other PMPS reactions could expand the economic viability of commercial peptides beyond current high-potency, low-volume therapeutics, with substantial green chemistry advantages

Functional Validation of an Alpha-Actinin-4 Mutation as a Potential Cause of an Aggressive Presentation of Adolescent Focal Segmental Glomerulosclerosis: Implications for Genetic Testing - Fig 1

<p>(A), histologic findings in the patient’s kidney biopsy. Periodic acid–Schiff (PAS) staining (magnification 200X) shows segmental glomerulosclerosis and interstitial fibrosis with multiple foci of microcystic tubular dilation. (B), Electron microscopy images shows glomerular podocyte foot processes effacement (arrows) (magnification 20000X). (C), serum creatinine and urine protein/creatinine ratio of the patient over a period of a year.</p

X-ray crystal structure of the PDZ1-IP target peptide chimera.

<p><b>A</b>: Amino acid sequence of the recombinant chimeric protein used for crystallization: N-terminal Gly-Ser dipeptide derived from the cloning vector (black), PDZ1 domain (residues 7–86 (PDZK1 numbering), green), partial interdomain segment (87–106, lies between the PDZ1 and PDZ2 domains, blue) and seven carboxy-terminal residues of IP (⁴¹¹IAACSLC⁴¹⁷, IP numbering, yellow). Regions of secondary structure (β strands and α helices) and the carboxylate-binding loop (CBL) are indicated above the sequence. <b>B</b>: Asymmetric unit showing the head-to-tail arrangement of two chimeric molecules. This figure was generated using POVScript <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053819#pone.0053819-Fenn1" target="_blank">[47]</a> using the color scheme in panel A.</p

Isothermal titration calorimetric analysis of the binding of a C-terminal peptide from IP to the PDZ1 domain of PDZK1.

<p>Recombinant wild-type PDZ1 domain (0.03 mM in 150 mM NaCl, 0.5 mM tris (2-carboxyethyl) phosphine, 25 mM Tris-pH 8.0) were placed in the titration cell and equilibrated at 20°C. A solution containing 1.0 mM of (A) the C-terminal nonapeptide from IP, KK⁴¹¹IAACSLC⁴¹⁷, or (B) the octapeptide K⁴⁰⁷SEAIAAC⁴¹⁴ corresponding to the C-terminal sequence of IP from which the last three amino acid –SLC are absent, or (C) the farnesylated-carboxy-methylated form of the same peptide (K⁴⁰⁷SEAIAAC⁴¹⁴⁾ were injected in 10 µl aliquots with an interval of 4 minutes between each addition to permit re-equilibration. Titration curves were analyzed and K_d values determined using ORIGIN 7.0 software.</p

Surface topology of PDZ1 and its bound target peptide.

<p>Surfaces of PDZ1 (gray) is shown bound to the IP target peptide (⁻⁶IAACSLC⁰, stick representation with the amino and carboxy-termini labeled “N” and “C”). The hydrophobic binding pocket accommodating the side chain of ⁰Cys can be seen as a deep cavity near the C-terminus of the target peptide. This figure was generated using PyMOL <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053819#pone.0053819-DeLano1" target="_blank">[49]</a>.</p

Steps involved in the isoprenylation and processing of the Prostacyclin Receptor.

<p>The prostacyclin receptor (IP) contains an evolutionary conserved ‘<i>CaaX</i> motif’ at its cytoplasmic carboxy-terminus, <i>e.g</i> corresponding to C⁴¹⁴SLC⁴¹⁷ of the mouse IP as shown. During its processing, (i) the IP undergoes isoprenylation through <i>thio-ether</i> attachment of a carbon (C)-15 farnesyl moiety to Cys⁴¹⁴ while subsequent (ii) proteolytic cleavage, or <i>aaX</i>ing, liberates the terminal ⁴¹⁵<i>SLC</i>⁴¹⁷ residues and (iii) end-stage carboxy-methylation of the nascent α-carboxy-group on Cys⁴¹⁴ generates the fully processed, mature IP in its farnesyl-Cys-carboxymethylated form. Herein, the interaction of peptides based on the mouse IP carboxy-terminus with PDZ domain 1 (PDZ1) or full length PDZK1 was investigated through isothermal titration calorimetry (ITC) where Peptide 1 is a nanopeptide containing the seven carboxy-terminal amino acids (KK⁴¹¹IAACSLC⁴¹⁷); Peptide 2 is an octapeptide corresponding to the carboxy-terminus of IP (K⁴⁰⁷SEAIAAC⁴¹⁴) devoid of the 3 terminal amino acids (-<i>aaX</i>/-⁴¹⁵<i>SLC</i>⁴¹⁷) which are proteolytically cleavage following farnesylation of the IP; Peptide 3 is identical to Peptide 2 (K⁴⁰⁷SEAIAAC⁴¹⁴) except that it was modified by the addition of a C-15 farnesyl group on the carboxy-terminal cysteine (Cys⁴¹⁴) and a carboxy-methyl group on the terminal -α-COOH, thereby representing the farnesyl-Cys-carboxymethyl ester form of the C-terminus of the IP.</p

Structure of the C-terminal IP target peptide binding to PDZ1.

<p><b>A</b>: Ribbon diagram showing the three dimensional structure of PDZ1 (residues 7–86, green with gray carboxylate-binding loop) and the bound C-terminus of IP (⁻⁶IAACSLC⁰, yellow) from an adjacent molecule in the asymmetric unit. Six β-strands (β1-β6), two α-helices (α1–α2) and carboxylate-binding loop (dark gray) are indicated. Vector derived residues have been omitted for clarity. <b>B</b>: Two-dimensional representation of interactions between PDZ1 (green) and the C-terminal IP target peptide (yellow). Hydrogen bonds are shown as dashed lines and hydrophobic interactions as arcs with radial spokes. This figure was generated using LIGPLOT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053819#pone.0053819-Wallace1" target="_blank">[48]</a>. <b>C</b>: Stereo representation of the ligand-binding groove of PDZ1 (green) and the IP target peptide (yellow). Oxygen, nitrogen and waters molecules are shown in red, dark blue and cyan, respectively. Sulfur atoms are colored in yellow. Hydrogen bonds are shown as dashed lines. The orientation is similar to that in panel A.</p

Structure determination and refinement statistics.

a<p>Values in parenthesis are for the highest resolution shell. R_sym = Σ|I-<i>|/Σ(I), where I is the observed integrated intensity, <i> is the average integrated intensity obtained from multiple measurements, and the summation is over all observable reflections. R_cryst = Σ||F_obs|-<i>k</i>|F_calc||/Σ|F_obs|, where F_obs and F_calc are the observed and calculates structure factors, respectively. R_free is calculated as R_cryst using 5% of the reflection chosen randomly and omitted from the refinement calculations. Bond lengths and angles are root-mean-square deviations from ideal values.</i></i></p