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Topology: a unique dimension in protein engineering

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  • ReceivedAug 17, 2017
  • AcceptedOct 12, 2017
  • PublishedDec 13, 2017

Abstract

Controlling protein topology has been a long standing challenge to go beyond their linear configuration defined by the translation mechanism of cellular machinery. In this mini-review, we focus on the topological diversity in proteins and review the major categories of protein topologies known to date, including branched/star proteins, circular proteins, lasso proteins, knotted proteins, and protein catenanes. The discovery of these topologically complex natural proteins and their synthetic pathways, the rational design and recombinant synthesis of artificial topological proteins and their biophysical studies, are summarized and discussed with regard to their general features and broad implications. The complexity of protein topology is recognized and the routes to diverse protein topologies are illustrated. We believe that topology engineering is an important way to modify protein properties without alternating their native sequences and shall bring in valuable dynamic features central to the creation of artificial protein machinery.


Funded by

National High Technology Research and Development Program of China(2015AA020941)

National Natural Science Foundation of China(21474003,91427304)

“1000 Plan(Youth)


Acknowledgment

This work was supported by the National High Technology Research and Development Program of China (2015AA020941), the National Natural Science Foundation of China (21474003, 91427304), and “1000 Plan (Youth)”.


Interest statement

The authors declare that they have no conflict of interest.


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  • Figure 1

    Typical chemical topologies, including linear, star, brush, circular, slipknot, knot (open or close), tadpole, lasso, (pseudo)rotaxane, and catenane (color online).

  • Figure 2

    Circular proteins. (a) Natural circular peptides, including AS-48 (PDB code: 1E68), RTD-1 (PDB code: 1HVZ), and kalata B1 (PDB code: 1KAL). (b) Traceless ligation methods, including split-intein technology, sortase-mediated ligation and butelase-mediated ligation. Adapted with permission from Refs. [23,24], copyright 2009 American Chemical Society and 2016 Macmillan Publishers Ltd., respectively. (c) Genetically encoded peptide-protein reactive pair enabled cyclization. Adapted with permission from Refs. [20,25], copyright 2013 American Chemical Society and 2014 Wiley-VCH, respectively (color online).

  • Figure 3

    Knotted protein topologies. (a) Line drawing of different knots. (b) Natural knotted proteins and their corresponding line drawings. From left to right: YbeA, trefoil (31), PDB code: 1NS5; ketol-acid reductoisomerase, figure-of-eight (41), PDB code: 3FR8; ubiquitin hydrolase UCH-L3, three-twist (52), PDB code: 1XD3; α-haloacid dehalogenase, stevedore knot (61), PDB code: 3BJX. Adapted with permission from Ref. [58], copyright 2016 Royal Society of Chemistry. (c) Mechanically tightening revealed a two-state unfolding pathway by majority of AFV3-109. Adapted with permission from Ref. [59], copyright 2014 American Chemical Society (color online).

  • Figure 4

    Lasso peptides. (a) Classification and line drawing of lasso peptides. (b) Typical lasso peptides in each category. From left to right, Class I, microcin J25, PDB code: 1Q71; Class II, BI-32169, PDB code: 3NJW; Class III, RP71955, PDB code: 1RPB. Adapted with permission from Ref. [70], copyright 2012 Royal Society of Chemistry. “Class IV”, LP2006, PDB code: 5JPL. Reprinted by permission from Ref. [71], copyright 2017 Macmillan Publishers Ltd. (color online).

  • Figure 5

    Synthesis of lasso peptide fusion proteins. Reprinted with permission from Ref. [76], copyright 2016 American Chemical Society (color online).

  • Figure 6

    Protein catenanes. (a) Line drawing of different types of links. (b) Natural protein catenanes, which is further divided into noncovalent catenanes (left) including (1) Cys168Ser variant of Prx III (PDB code:1ZYE), (2) class Ia RNR (PDB code: 4ERP), (3) CS2 hydrolase (PDB code: 3TEO), (4) RecR (PDB code: 1VDD) and covalent catenanes (right) including (5) the bacteriophage HK97 capsid and (6) Pyrobaculum aerophilum citrate synthase (PDB code: 2IBP). Adapted with permission from Ref. [58], copyright 2016 Royal Society of Chemistry (color online).

  • Figure 7

    Synthesis of protein catenanes. (a) Cellular synthesis of protein catenanes and mechanically interlocked tadpoles. Reprinted with permission from Ref. [21], copyright 2016 Wiley-VCH. (b) More complex topologies constructed by SpyX modules, including obligate dimers and star proteins. Adapted with permission from Ref. [19], copyright 2017 American Chemical Society (color online).

  • Figure 8

    (a) Cellular synthesis of protein catenanes with folded structural domains, including GFP and DHFR. (b) Effect of catenation on the stability, activity, and proteolytic resistance of the two folded proteins. Reprinted with permission from Ref. [22], copyright 2017 Wiley-VCH (color online).

  • Table 1   Summary of cyclization methods and the corresponding proteins

    Method

    In vivo cyclization

    Rate

    Traceless

    Internal sites

    Typical proteins

    Split intein technology

    Y

    Rapid

    Y

    N

    BLA [37], GFP [34], Bacillus subtilis family 11 xylanase [41], DnaB-N [42], E. coli glucose transporter [43]

    Sortase

    N

    Moderate

    Y

    N

    Cre recombinase [23], eGFP [23], human p97 [23], ubiquitin C-terminal hydrolase [23], human granulocyte colony-stimulating factor [44], human interferon-α [44], human erythropoietin [44], human growth hormone [40]

    Butelase

    N

    Rapid

    Y

    N

    GFP [45], human growth hormone [45], interleukin-1 receptor antagonist [45]

    SpyTag-SpyCatcher chemistry

    Y

    Rapid

    N

    Y

    BLA [25], DHFR [25], ELP [20], phytase [27], FLuc [38], lichenase [39]

    SnoopTag-SnoopCatcher chemistry

    Y

    Moderate

    N

    Y

    BLA [27]

  • Table 2   Summary of proteins with nontrivial topologies

    Topology

    Thermal stability

    Chemical stability

    Proteolytic stability

    Mechanical stability

    Examples

    Star proteins

    enhanced [60]

    3-arm star ELP [20], 4-arm star ELP [19,20]

    H-shape proteins

    H-shaped ELP [20]

    Circular proteins

    enhanced [25]

    enhanced [34]

    enhanced [37]

    AS-48 [46], circular BLA [25]

    Knotted proteins

    enhanced [69]

    enhanced [69]

    YbeA [64], 2ouf-knot based on HP0242 [67],

    Slipknotted proteins

    enhanced [57]

    enhanced [59]

    alkaline phosphatase [57]

    Lasso proteins

    enhanced [80]

    enhanced [72]

    MccJ25 [80], hybrid lasso peptide [79]

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