Sortase-mediated chemical protein synthesis reveals the bidentate binding of bisphosphorylated p62 with K63 diubiquitin† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc02937c

This work reports the first chemical synthesis of the phosphorylated p62 protein and reveals a bidentate binding model of bisphosphorylated p62.


A. Materials
Rink amide Resin and 2-Chlorotrityl Chloride Resin were purchased from NanKai Hecheng

B. HPLC, Mass spectrometry and FPLC
Analytical reverse phase HPLC (SHIMADZU, Prominence LC 20-AT) was used to monitor the purity of peptides and reaction progress with analytical columns (Welch XB C4 and Grace Vydac C8) with flow rate 1.0 mL/min. Semi-preparative RP-HPLC (SHIMADZU) was used to purify crude peptides and reaction products with semi preparative columns (Grace Vydac C4 and welch XB C18) with a flow rate of 6 mL/min and 10 mL/min, respectively.

C. Molecular biology and biochemistry
All primers for cloning were purchased from Ruibo Biotech (Beijing, China). Enzymes for cloning were purchased from New England Biolabs (England). LB medium was purchased from Baoruyi Biotech (Beijing, China).

D. Mass Spectrometry
High-resolution ESI mass spectra was measured by the Afilent 6210 Time of Flight Mass Spectrometer. Normal ESI mass spectra was measured by Bruker Daltonics Data Analysis 3.0.

Experimental Section
A. Peptide synthesis.

B. Cloning and Protein Expression.
The pET28a vector with human p62 gene inserted was a generous gift from Maojun Yang's Lab. Subsequently, various mutants and truncations of p62 are cloned by using primers according to the following table.
Primer Sequence, 5'-3' The expression and purification of K63 DiUb was based on previous reports. 1 C. General protocol for protein hydrazide ligation.

S5
The peptide hydrazine was dissolved in the Ligation Buffer (100 mM NaH 2 PO 4 pH 2.5, 6 M Gn-HCl) for a final concentration of 1 mM. After precooled and stirred in an ice-salt bath (-10℃), 10 eq of NaNO 2 was added to the reaction buffer for 30 min, then 40 eq of MPAA was added to the reaction mixture immediately to convert the peptide acyl azide into peptide thioester. Finally, 1.1eq of N-terminal cysteine peptide was added to the reaction mixture, and the pH was adjusted to 6.3. The reaction mixture was stirred overnight in room temperature to complete the ligation.
The reaction process was monitored and the product was further purified by RP-HPLC. 2

D. General protocol for Sortase A mediated peptide hydrazine expression.
The expressed segment 4 contains Sortase A cleavage site (LPETG) was purified as above methods. Then 1 eq of N terminal protein and 1 eq of Sortase A was added to the Converting Buffer (20 mM Tris pH 8, 150 mM NaCl, 10 mM CaCl 2 and 200 mM NH 2 NH 2 ) with a final concentration of 0.1 mM for segment 5. The reaction was stirred gently in 37℃ for 8 hours, the reaction process and the reaction product were monitored and further purified by RP-HPLC. 3

E. Protein folding and purification.
Synthesized proteins were lyophilized and then dissolved in a small amount of Folding Buffer

F. Circular diagram determination
CD spectra were recorded at 298 K on a Pistar π-180 CD spectrometer with a wavelength from 260 nm to 195 nm, and the concentrations of different p62 proteins were diluted to 0.1-0.2 mg/mL. Each sample was measured at least three times.

G. Surface plasmon resonance determination.
The binding force between all kinds of p62 and K63 DiUb were measured by Biacore T200 (GE Healthcare, Sweden) at 25℃. Synthetic phosphorylated p62 and expressed p62 as ligands were diluted to 0.03 mg/mL with ACE buffer, pH 4.5. Then ligand was covalently immobilized to a fresh CM5 sensor chip (GE Healthcare) immediately. The immobilization step was accomplished manually with a final response unit (RU) value of 400. The binding force S6 measurement was run at 30 uL/min in HBS-EP + buffer (GE Healthcare). To measure the binding affinity, K63 DiUb as analyte was diluted to at least 10 concentrations with HBS-EP + buffer using a double dilution method, and the concentration range was properly optimized for different substrates. The regeneration step was not needed. A variety of non-zero concentrations and a zero-concentration analyte were injected into the chip at a flow rate of 30 uL/min, and the contact and dissociation time were 60 seconds and 30 seconds respectively. The RU value was collected and all the experimental data was comprehensively analyzed by Evaluation Software (Biacore T200). Each sample was measured three times.

H. MS/MS identification of specific phosphorylation site.
For LC-MS/MS analysis of different site phosphorylated peptides, the peptide fragments were isolated by eluting with a 60 min gradient elution with a flow rate of 0.25 μL/min using a Thermo-Dionex Ultimate 3000 HPLC system, which was directly linked to the Thermo LTQ-Orbitrap Velos pro mass spectrometer. The MS/MS spectra was searched against p62 database in Proteome Discoverer (Vesion 1.4) searching algorithm. Analytical HPLC chromatogram and ESI mass spectra of purified MesNa thioester Segment 6. S11 S12 S13 Figure