Characterization Of The Substrate Specificity Of SUMO Protease And Its Engineering For Expanded Applications

SUMO protease(Ulp1)is a highly specific and active cysteine protease co m Monly used in industry and academia,which is often used to remove the N-ter minal SUMO protein(Smt3)fusion tag of a target protein during protein purification.Ulp1 cleaves the Smt3 protein tag by specifically recognizing the Smt3 protein structure to generate the final target protein with a natural N-ter minus.An interesting biochemical property of Ulpl is that it can efficiently recognize all 19 a mino acids except the Pro residue at the P1' site of its substrate.However,the biochemical mechanism of behind this characteristic was still unclear,and its non-recognition of Pro at P1' site also limits the use of Smt3 and Ulp1 in the protein fusion expression and purification.In this study,we studied two aspects of Ulp1: 1)perfor ming in-depth analysis of the substrate specificity of Ulp1 based on the Ulp1-Smt3 interaction structure,2)engineering Ulp1 according to the specificity of Ulp1 to enable it to cleave Smt3 with Pro at P1' site,thus expanding its applications in academia,industry,and medicine.First,we applied a new strategy based on the Yeast Endoplasmic reticulum Sequestration Screening(YESS)system to decipher the specific interaction of Ulpl against Smt3 by exploring the interaction interface of Ulp1-Smt3,the C-ter minal tail sequence of Smt3 and the P1-P2 positions of Smt3.We found that the interaction sites among the Ulp1-Smt3 interaction interface were functionally complementary,and a mino acid mutations in a single region did not significantly affect the recognition of Ulp1 against Smt3.Additionally,our studies on the C-ter minal tail sequence of Smt3 demonstrated that the last five a mino acid of Smt3 played an important role in the recognition of Smt3 by Ulp1.The deletion of the C-ter minal E-Q-I a mino acid resulted in a significantly decreased cleavage efficiency of Ulp1 against Smt3.Interestingly,it was found that the cleavage site of Ulp1 against Smt3 was not absolutely fixed,and the displacement of the cleavage site was based on the a mino acid sequence preference.Moreover,point mutation of P1-P2 sites indicated that Ulpl could recognize Ala/Gly at the P1 site,and Ala/Gly/Ser/Cys at the P2 site,indicating that the P1 site was significantly less tolerant than the P2 site.Combining the specificity and protein structure of Ulp1 against Smt3,it could be speculated that Trp448,Ser513,His514,Trp515 and Cys580 together formed a special and narrow substrate entry pocket in Ulpl to deter min the a mino acid preference at the P1-P2 sites of Smt3,in which the ?-? stacking force formed by the aromatic ring of the Trp448 side chain and the Gly-Gly peptide bond ring of P1-P2 sites fixed the orientation of the a mino acids at the P1-P2-P1' positions.Structural analysis suggested that the characteristic structure of the S1' substrate pocket of Ulp1 might result in the repression of the catalytic Cys580 by the side-chain of Pro residue at the P1' site.Therefore,in order to engineer Ulp1 to cleave the Gly Gly?Pro sequence,five a mino acids including Ser513,Leu533,Asn576,Gly577,and Tyr578 in the S1' substrate pocket was selected to construct a variant library,which was transformed into the YESS for variant analysis.After multiple rounds of cell sorting using flow cytometry,a few mutants with potential positive effects were obtained.Sequence analysis of these mutants revealed that: 1)Y578G mutation was critical for the structural changes of the Ulp1 S1' binding pocket,2)aromatic a mino acid preference at 533,576,and 577 sites was observed,and 3)deletion mutation at 513 site happened.Subsequently,we focused on the ZHR2-1 and ZMR3-10 do minant mutants,which achieved 50% and 90% cleavage efficiencies,respectively,of Ulp1 against the Smt3 with Gly Gly?Pro sequence.Taking ZHR2-1 as an example,the structural simulation of the ZLR2-1-Smt3 complex revealed that the structural change of the substrate entry pocket in Ulp1 was crucial for the proteolytic cleavage when Pro was the P1' residue.In the ZMR2-1 mutant,mutations in the Asn576,Gly577,and Tyr578 sites led to an open substrate entry pocket,causing a flexible orientation of the side chain group of Pro residue at the P1' site to enabling the catalytic Cys580 in Ulp1 could contact and cleave the peptide bond between P1 and P1' residue at the C-termus of Smt3.Among these mutations,the Y578 G mutation was particularly important,which could expand the S1' binding pocket space and release the steric repression of Arg446 to enable it to be pulled closer to Ser577 through the hydrogen bond interaction.The change of the spatial structure of Arg446 caused the displacement of the Trp448,which subsequently impaired the ?-? stacking force formed between the aromatic ring of the Trp448 side chain and the peptide bond ring of Gly-Gly sequence at the P1-P2 sites.It was worth noting that this changed the orientation of the Gly Gly Pro sequence in Smt3 orientation,which was further stabilized by the G576 Y mutation.In su m Mary,we analyzed the specific recognition and cleavage of Ulp1 against Smt3.Combining the structural simulation with high-throughput screening methods,we engineered Ulp1 to recognize and cleave Smt3 with Gly Gly?Pro sequence.These results prompted our understanding of Ulp1 and expanded its application.Moreover,this is also the first successful attempt in the directed evolution of proteases whose substrate is a protein rather than a short peptide.