Facilitating Subtiligase-Catalyzed Peptide Ligation Reactions by

Oct 16, 2018 - Chemistry Department and Center for Nucleic Acids Science and Technology, Carnegie Mellon University , Pittsburgh , Pennsylvania 15213 ...
0 downloads 0 Views 1MB Size
Download PDF
Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

pubs.acs.org/OrgLett

Facilitating Subtiligase-Catalyzed Peptide Ligation Reactions by Using Peptide Thioester Substrates Xiaohong Tan,*,† Renliang Yang,‡ and Chuan-Fa Liu*,‡ †

Downloaded via UNIV OF WINNIPEG on October 16, 2018 at 17:45:03 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Chemistry Department and Center for Nucleic Acids Science and Technology, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, United States ‡ Division of Chemical Biology and Biotechnology, School of Biological Sciences, Nanyang Technological University, Singapore 637551, Singapore S Supporting Information *

ABSTRACT: By facilitating formation of the acyl−enzyme intermediate, peptide thioesters can largely promote subtiligase-catalyzed ligation reactions over their ester counterparts. This offers a significant addition to the repertoire of enzymatic methods for protein chemical synthesis and modification.

P

by solid-phase peptide synthesis using Boc6,7 or Fmoc chemistry.8 For the initial attempt in proof of concept demonstration, we compared the subtiligase-catalyzed ligation reactions using one of its model peptide glycolate esters (AcHAAPF-ester-FG)3 and its thioester analogue derived from thioglycolate (Ac-HAAPF-thioester-FG). Both substrates reacted with a peptide nucleophile, H-AFA-NH2, respectively, catalyzed by subtiligase. Compared with Ac-HAAPF-ester-FG which required at least 65 min to complete the ligation reaction (Figure 2A), using a peptide thioester can greatly

rotein chemical modification or synthesis is extremely important for biomedical and biotechnological research, in which enzyme-catalyzed reactions play a unique role. For example, peptide ligase, as an enzyme which can catalyze the joining of two polypeptides by forming a new amide bond, has been frequently used in protein chemical synthesis or modification.1 One representative example of these enzymes is subtiligase, which is an engineered double mutant of subtilisin (Ser221Cys and Pro225Ala) developed by Wells and co-workers.2 With a Cys residue as its active site, subtiligase lacks the normal amidase activity of subtilisin and has weak hydrolase activity, but it retains significant esterase activity to catalyze the aminolysis of a suitable peptide ester for peptide ligation.2a−c Because of its strongly favoring aminolysis (thiolysis) over hydrolysis, we used subtiligase in preparing peptide thioesters3 and thioacids,4 in which subtiligase acts on the peptide ester substrate to generate an acyl−enzyme thioester intermediate which is further attacked by a nucleophile thiol group to give a thiolysis product.3,4 Based on this mechanism, we hypothesized that if the peptide ester is replaced by its peptide thioester analogy the acyl−enzyme thioester intermediate formation will be facilitated due to the very fast thiol−thioester exchange reaction,5 which in turn will improve the efficiencies of the subtiligase-catalyzed aminolysis reaction (Figure 1). Peptide thioesters are important building blocks in the semi or total synthesis of protein,6 and they can be readily prepared

Figure 2. HPLC monitoring of subtiligase-catalyzed ligation of HAFA-NH2 with (A) Ac-HAAPF-ester-FG-NH2 or (B) Ac-HAAPFthioester-FG-NH2. Peak a: ligation product, Ac-HAAPFAFA-NH2. ESI-MS analysis: (m/z) [M + H]+ found: 872.3, MW calcd: 871.4 (isotopic); peak b: Ac-HAAPF-thioester-FG-NH2; and peak c: AcHAAPF-ester-FG-NH2. The reaction buffers were 0.12 M bicine (pH 8) containing 10 mM TCEP, 0.2 mM peptide thioester or ester substrate, 5 mM H-AFA-NH2, and 0.25 μM subtiligase.

Figure 1. Using peptide thioesters as substrates in subtiligasemediated peptide ligation. © XXXX American Chemical Society

Received: August 27, 2018

A

DOI: 10.1021/acs.orglett.8b02747 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters shorten the reaction time to 3 min (Figure 2B). In addition, no significant hydrolysis or uncatalyzed aminolysis of the peptide thioester substrate was observed in such a short time. This significant improvement indicates that the peptide thioester is a much more active substrate than the peptide ester in subtiligase-catalyzed reactions. Next, we tested whether peptide thioesters can facilitate the subtiligase-catalyzed ligation using its unfavorable peptide substrate sequences. For example, it was reported that Ile or Gly should be avoided at the P1 position of a peptide ester.2a Therefore, we first synthesized a peptide ester (Ac-LVKEIester-FG) and its peptide thioester analogue (Ac-LVKEIthioester-FG), which were ligated with the same nucleophile (H-AFA-NH2). As shown in Figure 3A, the ligation reaction

Table 1. Comparison of Kinetic Constants of the Subtiligase-Catalyzed Ligations for Peptide Ester and Thioester Substratesa substrates

KM (mM)

kcat (min−1)

Ac-HAAPF-ester-FG Ac-HAAPF-thioester-FG Ac-KPGTVA-ester-FG Ac-KPGTVA-thioester-FG Ac-LVKEI-ester-FG Ac-LVKEI-thioester-FG

0.555 0.147 1.092 2.390 0.132 0.273

336.1 2198 57.97 2857 8.333 196.1

kcat/KM (min−1 mM−1) 6.06 1.49 53.1 1.20 63.3 7.18

× 102 × 104 × 103 × 102

a

Kinetic constants were calculated by using the Lineweaver−Burk double-reciprocal plot. See Supporting Information and Figure S1 for details.

thioester-FG was 22-fold higher than that of Ac-KPGTVAester-FG. For the third group, containing a undesirable Ile residue at the P1 position for subtiligase,2a the reaction efficiency of Ac-LVKEI-thioester-FG was enhanced 11-fold compared to Ac-LVKEI-ester-FG. The latter two-group peptide esters were expected to be subtiligase-undesirable substrates, which were confirmed by their much smaller kcat/ KM values compared to the model ester substrate Ac-HAAPFglc-FG (Table 1). Moreover, we observed that the latter two peptide thioesters have even higher KM values compared to their peptide ester analogues (Table 1), indicating that the increased catalytic efficiencies (kcat/KM) only result from increased kcat values, especially for Ac-KPGTVA-X-FG which has a 50-fold enhanced kcat value compared with its peptide ester analogue. These data suggest that the use of thioester substrates does not improve the KM but directly enhances the enzyme efficiency in peptide ligation. This is due to the much higher efficiency of the thiol−thioester exchange reaction than the thiol−ester exchange reaction in the formation of the acylenzyme intermediate. To further understand why peptide thioesters are better substrates than esters, we examined the ligation reactions using another peptide nucleophile (H-GGLG-NH2), which is predicted to be a very poor substrate for subtiligase.2a We observed that all ligations were very slow, and there was no significant difference between using ester and thioester as substrates (data not show). These data indicate that replacing peptide ester with thioester does not affect the procedure when nucleophile attacks the acyl-enzyme thioester intermediate, suggesting a possible mechanism in which peptide thioester substrates may facilitate the faster formation of acyl-enzyme thioester intermediate than peptide ester substrates. Next we attempted to optimize the subtiligase-catalyzed protein modification by using peptide thioesters as substrates. Human IgG Fc region (huFc) was chosen as the modification target. A short peptide sequence AFA, which is an optimized peptide nucleophile of subtiligase,2a was genetically introduced into the N-terminus of huFc. Then this modified huFc (AFAhuFc) was conjugated with Ac-HAAPF-ester-FG or AcHAAPF-thioester-FG by subtiligase. It was expected that a peptide segment (Ac-HAAPF) will be added onto the Ntermimus of the protein to form Ac-HAAPFAFA-huFc, whose MALDI-MS value is 572 Da more than that of AFA-huFc; therefore, MALDI-MS analysis can be utilized to monitor the reaction procedures. As shown in Figure 4A, after only 1 min, Ac-HAAPF-thioester-FG converted more than 50% of AFAhuFc to Ac-HAAPFAFA-huFc. After 6 min, the yield was

Figure 3. Fraction of substrates converted to either ligation or hydrolysis product as a function of time by subtiligase: (A) AcLVKEI-ester-FG and (B) Ac-LVKEI-thioester-FG. The reaction buffers were 0.12 M bicine (pH 8) containing 10 mM TCEP, 5 mM H-AFA-NH2, 0.25 μM subtiligase, and 0.2 mM peptide substrate. Yields were calculated by using normalized HPLC absorption peak areas at λ = 220 nm.

using Ac-LVKEI-ester-FG was slow. After 390 min, only 62% ester substrate formed the ligation product (Ac-LVKEIAFANH2), while 18% ester substrate was hydrolyzed (Ac-LVKEIOH). However, when we conducted the ligation reaction using its peptide thioester analogue (Ac-LVKEI-thioester-FG), after 80 min, almost all of the thioester substrate was converted to ligation product without any observable hydrolysis product, suggesting again that subtiligase prefers peptide thioester to ester substrates in ligation reactions. Second, we synthesized the other subtiligase-unfavorable peptide ester (Ac-LVKEG-ester-FG) and its peptide thioester analogue (Ac-LVKEG-thioester-FG), which were ligated with the same nucleophile (H-AFA-NH2). For the ester, there was no observable ligation product since subtiligase completely hydrolyzed it to Ac-LVKEG-OH, but 10% of the peptide thioester substrate was converted to ligation product (AcLVKEGAFA-NH2) after 12 h (data not shown). These data demonstrated that compared with peptide esters peptide thioesters can overcome the restriction of substrate specificity of subtiligase as much as possible and facilitate peptide ligation reactions. Furthermore, as shown in Table 1, we compared kinetic parameters of peptide thioester and ester substrates in subtiligase-catalyzed ligation reactions. For the first group, containing the model peptide substrate sequence, the kcat/KM value of Ac-HAAPF-thioester-FG was enhanced 24-fold compared to that of Ac-HAAPF-ester-FG, contributing from both of the higher kcat value and lower KM value. For the second group, containing an undesirable Gly residue at the P4 position for subtiligase,2a the kcat/KM value of Ac-KPGTVAB

DOI: 10.1021/acs.orglett.8b02747 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters

Healthcare). According to nonreduced SDS-PAGE analysis, the modified AFA-huFc refolded well to form a dimer as AFAhuFc (Figure 5B). The high conversion rate and preservation of protein activity clearly illustrate the factual applications of our optimized subtiligase-catalyzed protein modifications. In conclusion, we have proven that peptide thioesters are much better substrates than peptide esters in subtiligasecatalyzed ligation reactions. We demonstrated the high efficiency of using peptide thioesters in both peptide and protein ligations. We also revealed that peptide thioesters can partially overcome the restriction of substrate specific for subtiligase, expanding the diversity of peptide substrates. In a recent paper, Cole and co-workers used protein thioesters as subtiligase substrates for protein modifications.1c However, to the best of our knowledge, our study is the first report to directly compare the ligation efficiency of using peptide esters and thioesters in a subtiligase-mediated ligation reaction. By measuring kinetic parameters and collecting other data, we provided evidence to support that peptide thioesters can facilitate the formation of an acyl-enzyme thioester intermediate much more quickly than peptide esters. Thioesters have excellent stability and are widely used as essential building blocks in peptide ligation.6,7,9 Using peptide thioesters can be a very powerful approach for subtiligase-based protein chemical synthesis and modification, with the potentials to be used for other cysteine-based peptide ligases.

Figure 4. MALDI-MS analysis of ligation between HuFc and (A) AcHAAPF-thioester-FG-NH2 or (B) Ac-HAAPF-ester-FG-NH2. The reaction buffers were 0.15 M bicine (pH 9) containing 10 mM TCEP, 0.5 mg/mL of huFc, 0.2 mM peptide substrate, and 0.15 μM subtiligase. Yields were calculated by using normalized MALDI-MS peak areas.

above 90%. However, the reaction between AFA-huFc and AcHAAPF-ester-FG was relatively slow; e.g., it required more than 20 min to convert 50% of AFA-huFc into the product. These results agree well with our previous peptide data, indicating the priorities of using peptide thioester substrates in subtiligase-catalyzed protein bioconjugation. To further prove that our method can be generalizable to other peptides, especially those containing unnatural residues, we performed the bioconjugation of angiotensin II (H-SarRVYIHPA-OH) to AFA-huFc by its thioester (H-SarRVYIHPA-thioester-FG-NH2). As shown in Figure 5A, the

■

ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b02747. Experimental details (PDF)

■

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: cfl[email protected]. ORCID

Xiaohong Tan: 0000-0001-7272-4292 Chuan-Fa Liu: 0000-0001-7433-2081 Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

■

Figure 5. (A) MALDI-MS analysis of ligation between HuFc and angiotensin II (SarAla8). The reaction buffers were 0.15 M bicine (pH 9) containing 10 mM TCEP, 0.5 mg/mL of huFc, 0.2 mM peptide substrate, and 0.5 μM subtiligase. (B) SDS-PAGE gel of 1) protein marker; 2) dimerization of original HuFC; 3) dimerization of biconjugated HuFc (H-Sar-RVYIHPAAFA-HuFc).

ACKNOWLEDGMENTS The authors thank the Ministry of Education of Singapore for financial support (MOE 2016-T3-1-003) as well as Nanyang Technological University. Xiaohong Tan thanks the postdoctoral fellowship supported from the David Scaife Family Charitable Foundation.

ligation reaction was completed within 2 h, and actually after 1 h more than 70% of the ligation reaction between AFA-huFC and the peptide thioester was completed. Next, we asked whether the N-terminal conjugated huFc can still form a disulfide-bonded dimer. The angiotensin II-conjugated AFAhuFc (H-Sar-RVYIHPAAFA-huFc) was suspended into the refolding buffer and purified by MAbselect Xtra column (GE

■

REFERENCES

(1) (a) Bi, X.; Yin, J.; Nguyen, G. K. T.; Rao, C.; Halim, N. B. A.; Hemu, X.; Tam, J. P.; Liu, C.-F. Angew. Chem. 2017, 129, 7930. (b) Nguyen, G. K. T.; Wang, S.; Qiu, Y.; Hemu, X.; Lian, Y.; Tam, J. P. Nat. Chem. Biol. 2014, 10, 732. (c) Henager, S. H.; Chu, N.; Chen,

C

DOI: 10.1021/acs.orglett.8b02747 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters Z.; Bolduc, D.; Dempsey, D. R.; Hwang, Y.; Wells, J. A.; Cole, P. A. Nat. Methods 2016, 13, 925. (d) Jackson, D. Y.; Burnier, J.; Quan, C.; Stanley, M.; Tom, J.; Wells, J. A. Science 1994, 266, 243. (e) Lu, X.; He, S.-J.; Cheng, W.-M.; Shi, J. Chin. Chem. Lett. 2018, 29, 1001. (f) Schmidt, M.; Toplak, A.; Quaedflieg, P. J. L. M.; Nuijens, T. Curr. Opin. Chem. Biol. 2017, 38, 1. (2) (a) Abrahmsen, J. T. L.; Burnier, J.; Butcher, K. A.; Kossiakoff, A.; Wells, J. A. Biochemistry 1991, 30, 4151. (b) Chang, T. K.; Jackson, D. Y.; Burnier, J. P.; Wells, J. A. Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 12544. (c) Braisted, A. C.; Judice, J. K.; Wells, J. A. Methods Enzymol. 1997, 289, 298. (3) Tan, X. H.; Wirjo, A.; Liu, C. F. ChemBioChem 2007, 8, 1512. (4) Tan, X. H.; Yang, R.; Wirjo, A.; Liu, C.-F. Tetrahedron Lett. 2008, 49, 2891. (5) Tan, X. H.; Zhang, X.; Yang, R.; Liu, C.-F. ChemBioChem 2008, 9, 1052. (6) (a) Muir, T. W.; Dawson, P. E.; Kent, S. B. H. Methods Enzymol. 1997, 289, 266. (b) Aimoto, S. Biopolymers 1999, 51, 247. (7) (a) Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. Proc. Natl. Acad. Sci. U. S. A. 1999, 96, 10068. (b) Tam, J. P.; Lu, Y. A.; Liu, C.F.; Shao, J. Proc. Natl. Acad. Sci. U. S. A. 1995, 92, 12485. (8) (a) Li, X.; Kawakami, T.; Aimoto, S. Tetrahedron Lett. 1998, 39, 8669. (b) Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc. 1999, 121, 11369. (c) Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 1999, 121, 11684. (d) Brask, J.; Albericio, F.; Jensen, K. J. Org. Lett. 2003, 5, 2951. (e) Ohta, Y.; Itoh, S.; Shigenaga, A.; Shintaku, S.; Fujii, N.; Otaka, A. Org. Lett. 2006, 8, 467. (9) Bracher, P. J.; Snyder, P. W.; Bohall, B. R.; Whitesides, G. M. Origins Life Evol. Biospheres 2011, 41, 399.

D

DOI: 10.1021/acs.orglett.8b02747 Org. Lett. XXXX, XXX, XXX−XXX