Circular permutation in proteins

File:Concanavalin A vs Lectin.png (left), from the Protein Data Bank ({{PDB|3cna}}), and peanut lectin (right), from {{PDB|2pel}}, which is homologous to favin. The termini of the proteins are highlighted by blue and green spheres, and the sequence of residues is indicated by the gradient from blue (N-terminus) to green (C-terminus). The 3D fold of the two proteins is highly similar; however, the N- and C- termini are located on different positions of the protein.]]

In 1979, Bruce Cunningham and his colleagues discovered the first instance of a circularly permuted protein in nature.{{cite journal | vauthors = Cunningham BA, Hemperly JJ, Hopp TP, Edelman GM | title = Favin versus concanavalin A: Circularly permuted amino acid sequences | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 76 | issue = 7 | pages = 3218–22 | date = July 1979 | pmid = 16592676 | pmc = 383795 | doi = 10.1073/pnas.76.7.3218 | bibcode = 1979PNAS...76.3218C | doi-access = free }} After determining the peptide sequence of the lectin protein favin, they noticed its similarity to a known protein – concanavalin A – except that the ends were circularly permuted. Later work confirmed the circular permutation between the pair{{cite journal | vauthors = Einspahr H, Parks EH, Suguna K, Subramanian E, Suddath FL | title = The crystal structure of pea lectin at 3.0-A resolution | journal = The Journal of Biological Chemistry | volume = 261 | issue = 35 | pages = 16518–27 | date = December 1986 | doi = 10.1016/S0021-9258(18)66597-4 | pmid = 3782132 | doi-access = free }} and showed that concanavalin A is permuted post-translationally{{cite journal | vauthors = Carrington DM, Auffret A, Hanke DE | title = Polypeptide ligation occurs during post-translational modification of concanavalin A | journal = Nature | volume = 313 | issue = 5997 | pages = 64–7 | year = 1985 | pmid = 3965973 | doi = 10.1038/313064a0 | bibcode = 1985Natur.313...64C | s2cid = 4359482 }} through cleavage and an unusual protein ligation.

After the discovery of a natural circularly permuted protein, researchers looked for a way to emulate this process. In 1983, David Goldenberg and Thomas Creighton were able to create a circularly permuted version of a protein by chemically ligating the termini to create a cyclic protein, then introducing new termini elsewhere using trypsin.{{cite journal | vauthors = Goldenberg DP, Creighton TE | title = Circular and circularly permuted forms of bovine pancreatic trypsin inhibitor | journal = Journal of Molecular Biology | volume = 165 | issue = 2 | pages = 407–13 | date = April 1983 | pmid = 6188846 | doi = 10.1016/S0022-2836(83)80265-4 }} In 1989, Karolin Luger and her colleagues introduced a genetic method for making circular permutations by carefully fragmenting and ligating DNA.{{cite journal | vauthors = Luger K, Hommel U, Herold M, Hofsteenge J, Kirschner K | title = Correct folding of circularly permuted variants of a beta alpha barrel enzyme in vivo | journal = Science | volume = 243 | issue = 4888 | pages = 206–10 | date = January 1989 | pmid = 2643160 | doi = 10.1126/science.2643160 | bibcode = 1989Sci...243..206L }} This method allowed for permutations to be introduced at arbitrary sites.

Despite the early discovery of post-translational circular permutations and the suggestion of a possible genetic mechanism for evolving circular permutants, it was not until 1995 that the first circularly permuted pair of genes were discovered. Saposins are a class of proteins involved in sphingolipid catabolism and antigen presentation of lipids in humans. Chris Ponting and Robert Russell identified a circularly permuted version of a saposin inserted into plant aspartic proteinase, which they nicknamed swaposin.{{cite journal | vauthors = Ponting CP, Russell RB | title = Swaposins: circular permutations within genes encoding saposin homologues | journal = Trends in Biochemical Sciences | volume = 20 | issue = 5 | pages = 179–80 | date = May 1995 | pmid = 7610480 | doi = 10.1016/S0968-0004(00)89003-9 }} Saposin and swaposin were the first known case of two natural genes related by a circular permutation.

Hundreds of examples of protein pairs related by a circular permutation were subsequently discovered in nature or produced in the laboratory. As of February 2012, the Circular Permutation Database{{cite web |url=http://sarst.life.nthu.edu.tw/cpdb/ |title=Circular Permutation Database |first1=Wei-Cheng |last1=Lo |first2=Chi-Ching |last2=Lee |first3=Che-Yu |last3=Lee |first4=Ping-Chiang |last4=Lyu | name-list-style = vanc |publisher=Institute of Bioinformatics and Structural Biology, National Tsing Hua University |access-date=16 February 2012}} contains 2,238 circularly permuted protein pairs with known structures, and many more are known without structures.{{cite journal | vauthors = Lo WC, Lee CC, Lee CY, Lyu PC | title = CPDB: a database of circular permutation in proteins | journal = Nucleic Acids Research | volume = 37 | issue = Database issue | pages = D328–32 | date = January 2009 | pmid = 18842637 | pmc = 2686539 | doi = 10.1093/nar/gkn679 }} The CyBase database collects proteins that are cyclic, some of which are permuted variants of cyclic wild-type proteins.{{cite journal | vauthors = Kaas Q, Craik DJ | title = Analysis and classification of circular proteins in CyBase | journal = Biopolymers | volume = 94 | issue = 5 | pages = 584–91 | year = 2010 | pmid = 20564021 | doi = 10.1002/bip.21424 | doi-access = free }} SISYPHUS is a database that contains a collection of hand-curated manual alignments of proteins with non-trivial relationships, several of which have circular permutations.{{cite journal | vauthors = Andreeva A, Prlić A, Hubbard TJ, Murzin AG | title = SISYPHUS--structural alignments for proteins with non-trivial relationships | journal = Nucleic Acids Research | volume = 35 | issue = Database issue | pages = D253–9 | date = January 2007 | pmid = 17068077 | pmc = 1635320 | doi = 10.1093/nar/gkl746 }}

There are two main models that are currently being used to explain the evolution of circularly permuted proteins: permutation by duplication and fission and fusion. The two models have compelling examples supporting them, but the relative contribution of each model in evolution is still under debate.{{cite journal | vauthors = Weiner J, Bornberg-Bauer E | title = Evolution of circular permutations in multidomain proteins | journal = Molecular Biology and Evolution | volume = 23 | issue = 4 | pages = 734–43 | date = April 2006 | pmid = 16431849 | doi = 10.1093/molbev/msj091 | doi-access = free }} Other, less common, mechanisms have been proposed, such as "cut and paste"{{cite journal | vauthors = Bujnicki JM | title = Sequence permutations in the molecular evolution of DNA methyltransferases | journal = BMC Evolutionary Biology | volume = 2 | issue = 1 | pages = 3 | date = March 2002 | pmid = 11914127 | pmc = 102321 | doi = 10.1186/1471-2148-2-3 | doi-access = free }} or "exon shuffling".

File:Permutation by Duplication.svg

The earliest model proposed for the evolution of circular permutations is the permutation by duplication mechanism. In this model, a precursor gene first undergoes a duplication and fusion to form a large tandem repeat. Next, start and stop codons are introduced at corresponding locations in the duplicated gene, removing redundant sections of the protein.

One surprising prediction of the permutation by duplication mechanism is that intermediate permutations can occur. For instance, the duplicated version of the protein should still be functional, since otherwise evolution would quickly select against such proteins. Likewise, partially duplicated intermediates where only one terminus was truncated should be functional. Such intermediates have been extensively documented in protein families such as DNA methyltransferases.{{cite journal | vauthors = Jeltsch A | title = Circular permutations in the molecular evolution of DNA methyltransferases | journal = Journal of Molecular Evolution | volume = 49 | issue = 1 | pages = 161–4 | date = July 1999 | pmid = 10368444 | doi = 10.1007/pl00006529 | bibcode = 1999JMolE..49..161J | s2cid = 24116226 }}

File:Saposin Swaposin.svg

An example for permutation by duplication is the relationship between saposin and swaposin. Saposins are highly conserved glycoproteins, approximately 80 amino acid residues long and forming a four alpha helical structure. They have a nearly identical placement of cysteine residues and glycosylation sites. The cDNA sequence that codes for saposin is called prosaposin. It is a precursor for four cleavage products, the saposins A, B, C, and D. The four saposin domains most likely arose from two tandem duplications of an ancestral gene.{{cite journal | vauthors = Hazkani-Covo E, Altman N, Horowitz M, Graur D | title = The evolutionary history of prosaposin: two successive tandem-duplication events gave rise to the four saposin domains in vertebrates | journal = Journal of Molecular Evolution | volume = 54 | issue = 1 | pages = 30–4 | date = January 2002 | pmid = 11734895 | doi = 10.1007/s00239-001-0014-0 | bibcode = 2002JMolE..54...30H | s2cid = 7402721 }} This repeat suggests a mechanism for the evolution of the relationship with the plant-specific insert (PSI). The PSI is a domain exclusively found in plants, consisting of approximately 100 residues and found in plant aspartic proteases.{{cite journal | vauthors = Guruprasad K, Törmäkangas K, Kervinen J, Blundell TL | title = Comparative modelling of barley-grain aspartic proteinase: a structural rationale for observed hydrolytic specificity | journal = FEBS Letters | volume = 352 | issue = 2 | pages = 131–6 | date = September 1994 | pmid = 7925961 | doi = 10.1016/0014-5793(94)00935-X | s2cid = 32524531 | doi-access = free | bibcode = 1994FEBSL.352..131G }} It belongs to the saposin-like protein family (SAPLIP) and has the N- and C- termini "swapped", such that the order of helices is 3-4-1-2 compared with saposin, thus leading to the name "swaposin".{{cite journal | vauthors = Bruhn H | title = A short guided tour through functional and structural features of saposin-like proteins | journal = The Biochemical Journal | volume = 389 | issue = Pt 2 | pages = 249–57 | date = July 2005 | pmid = 15992358 | pmc = 1175101 | doi = 10.1042/BJ20050051 }}

File:Fission-fusion (genetics).svg

Another model for the evolution of circular permutations is the fission and fusion model. The process starts with two partial proteins. These may represent two independent polypeptides (such as two parts of a heterodimer), or may have originally been halves of a single protein that underwent a fission event to become two polypeptides.

The two proteins can later fuse together to form a single polypeptide. Regardless of which protein comes first, this fusion protein may show similar function. Thus, if a fusion between two proteins occurs twice in evolution (either between paralogues within the same species or between orthologues in different species) but in a different order, the resulting fusion proteins will be related by a circular permutation.

Evidence for a particular protein having evolved by a fission and fusion mechanism can be provided by observing the halves of the permutation as independent polypeptides in related species, or by demonstrating experimentally that the two halves can function as separate polypeptides.{{cite journal | vauthors = Lee J, Blaber M | title = Experimental support for the evolution of symmetric protein architecture from a simple peptide motif | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 1 | pages = 126–30 | date = January 2011 | pmid = 21173271 | pmc = 3017207 | doi = 10.1073/pnas.1015032108 | bibcode = 2011PNAS..108..126L | doi-access = free }}

File:Transhydrogenase Circular Permutations.svg, the three domains are arranged sequentially. In the bacteria E. coli, Rb. capsulatus, and R. rubrum, the transhydrogenase consists of two or three subunits. Finally, transhydrogenase from the protist E. tenella consists of a single subunit that is circularly permuted relative to cattle transhydrogenase.{{cite journal | vauthors = Hatefi Y, Yamaguchi M | title = Nicotinamide nucleotide transhydrogenase: a model for utilization of substrate binding energy for proton translocation | journal = FASEB Journal | volume = 10 | issue = 4 | pages = 444–52 | date = March 1996 | pmid = 8647343 | doi = 10.1096/fasebj.10.4.8647343 | doi-access = free | s2cid = 21898930 }}]]

An example for the fission and fusion mechanism can be found in nicotinamide nucleotide transhydrogenases. These are membrane-bound enzymes that catalyze the transfer of a hydride ion between NAD(H) and NADP(H) in a reaction that is coupled to transmembrane proton translocation. They consist of three major functional units (I, II, and III) that can be found in different arrangement in bacteria, protozoa, and higher eukaryotes. Phylogenetic analysis suggests that the three groups of domain arrangements were acquired and fused independently.

The two evolutionary models mentioned above describe ways in which genes may be circularly permuted, resulting in a circularly permuted mRNA after transcription. Proteins can also be circularly permuted via post-translational modification, without permuting the underlying gene. Circular permutations can happen spontaneously through autocatalysis, as in the case of concanavalin A.{{cite journal | vauthors = Bowles DJ, Pappin DJ | title = Traffic and assembly of concanavalin A | journal = Trends in Biochemical Sciences | volume = 13 | issue = 2 | pages = 60–4 | date = February 1988 | pmid = 3070848 | doi = 10.1016/0968-0004(88)90030-8 }} Alternately, permutation may require restriction enzymes and ligases.

Many proteins have their termini located close together in 3D space.{{cite journal | vauthors = Thornton JM, Sibanda BL | title = Amino and carboxy-terminal regions in globular proteins | journal = Journal of Molecular Biology | volume = 167 | issue = 2 | pages = 443–60 | date = June 1983 | pmid = 6864804 | doi = 10.1016/S0022-2836(83)80344-1 }}{{cite journal | vauthors = Yu Y, Lutz S | title = Circular permutation: a different way to engineer enzyme structure and function | journal = Trends in Biotechnology | volume = 29 | issue = 1 | pages = 18–25 | date = January 2011 | pmid = 21087800 | doi = 10.1016/j.tibtech.2010.10.004 }} Because of this, it is often possible to design circular permutations of proteins. Today, circular permutations are generated routinely in the lab using standard genetics techniques. Although some permutation sites prevent the protein from folding correctly, many permutants have been created with nearly identical structure and function to the original protein.

The motivation for creating a circular permutant of a protein can vary. Scientists may want to improve some property of the protein, such as:

• Reduce proteolytic susceptibility. The rate at which proteins are broken down can have a large impact on their activity in cells. Since termini are often accessible to proteases, designing a circularly permuted protein with less-accessible termini can increase the lifespan of that protein in the cell.{{cite journal | vauthors = Whitehead TA, Bergeron LM, Clark DS |author3-link=Douglas S. Clark | title = Tying up the loose ends: circular permutation decreases the proteolytic susceptibility of recombinant proteins | journal = Protein Engineering, Design & Selection | volume = 22 | issue = 10 | pages = 607–13 | date = October 2009 | pmid = 19622546 | doi = 10.1093/protein/gzp034 | doi-access = free }}
• Improve catalytic activity. Circularly permuting a protein can sometimes increase the rate at which it catalyzes a chemical reaction, leading to more efficient proteins.{{cite journal | vauthors = Cheltsov AV, Barber MJ, Ferreira GC | title = Circular permutation of 5-aminolevulinate synthase. Mapping the polypeptide chain to its function | journal = The Journal of Biological Chemistry | volume = 276 | issue = 22 | pages = 19141–9 | date = June 2001 | pmid = 11279050 | pmc = 4547487 | doi = 10.1074/jbc.M100329200 | doi-access = free }}
• Alter substrate or ligand binding. Circularly permuting a protein can result in the loss of substrate binding, but can occasionally lead to novel ligand binding activity or altered substrate specificity.{{cite journal | vauthors = Qian Z, Lutz S | title = Improving the catalytic activity of Candida antarctica lipase B by circular permutation | journal = Journal of the American Chemical Society | volume = 127 | issue = 39 | pages = 13466–7 | date = October 2005 | pmid = 16190688 | doi = 10.1021/ja053932h }} (primary source)
• Improve thermostability. Making proteins active over a wider range of temperatures and conditions can improve their utility.{{cite journal | vauthors = Topell S, Hennecke J, Glockshuber R | title = Circularly permuted variants of the green fluorescent protein | journal = FEBS Letters | volume = 457 | issue = 2 | pages = 283–9 | date = August 1999 | pmid = 10471794 | doi = 10.1016/S0014-5793(99)01044-3 | bibcode = 1999FEBSL.457..283T | s2cid = 43085373 }} (primary source)

Alternately, scientists may be interested in properties of the original protein, such as:

• Fold order. Determining the order in which different parts of a protein fold is challenging due to the extremely fast time scales involved. Circularly permuted versions of proteins will often fold in a different order, providing information about the folding of the original protein.{{cite journal | vauthors = Viguera AR, Serrano L, Wilmanns M | title = Different folding transition states may result in the same native structure | journal = Nature Structural Biology | volume = 3 | issue = 10 | pages = 874–80 | date = October 1996 | pmid = 8836105 | doi = 10.1038/nsb1096-874 | s2cid = 11542397 }} (primary source){{cite journal | vauthors = Capraro DT, Roy M, Onuchic JN, Jennings PA | title = Backtracking on the folding landscape of the beta-trefoil protein interleukin-1beta? | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 105 | issue = 39 | pages = 14844–8 | date = September 2008 | pmid = 18806223 | pmc = 2567455 | doi = 10.1073/pnas.0807812105 | bibcode = 2008PNAS..10514844C | doi-access = free }}{{cite journal | vauthors = Zhang P, Schachman HK | title = In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: implications for protein folding and assembly | journal = Protein Science | volume = 5 | issue = 7 | pages = 1290–300 | date = July 1996 | pmid = 8819162 | pmc = 2143468 | doi = 10.1002/pro.5560050708 }} (primary source)
• Essential structural elements. Artificial circularly permuted proteins can allow parts of a protein to be selectively deleted. This gives insight into which structural elements are essential or not.{{cite journal | vauthors = Huang YM, Nayak S, Bystroff C | title = Quantitative in vivo solubility and reconstitution of truncated circular permutants of green fluorescent protein | journal = Protein Science | volume = 20 | issue = 11 | pages = 1775–80 | date = November 2011 | pmid = 21910151 | pmc = 3267941 | doi = 10.1002/pro.735 }} (primary source)
• Modify quaternary structure. Circularly permuted proteins have been shown to take on different quaternary structure than wild-type proteins.{{cite journal | vauthors = Beernink PT, Yang YR, Graf R, King DS, Shah SS, Schachman HK | title = Random circular permutation leading to chain disruption within and near alpha helices in the catalytic chains of aspartate transcarbamoylase: effects on assembly, stability, and function | journal = Protein Science | volume = 10 | issue = 3 | pages = 528–37 | date = March 2001 | pmid = 11344321 | pmc = 2374132 | doi = 10.1110/ps.39001 }}
• Find insertion sites for other proteins. Inserting one protein as a domain into another protein can be useful. For instance, inserting calmodulin into green fluorescent protein (GFP) allowed researchers to measure the activity of calmodulin via the fluorescence of the split-GFP.{{cite journal | vauthors = Baird GS, Zacharias DA, Tsien RY | title = Circular permutation and receptor insertion within green fluorescent proteins | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 96 | issue = 20 | pages = 11241–6 | date = September 1999 | pmid = 10500161 | pmc = 18018 | doi = 10.1073/pnas.96.20.11241 | bibcode = 1999PNAS...9611241B | doi-access = free }} Regions of GFP that tolerate the introduction of circular permutation are more likely to accept the addition of another protein while retaining the function of both proteins.
• Design of novel biocatalysts and biosensors. Introducing circular permutations can be used to design proteins to catalyze specific chemical reactions,{{cite journal | vauthors = Turner NJ | title = Directed evolution drives the next generation of biocatalysts | journal = Nature Chemical Biology | volume = 5 | issue = 8 | pages = 567–73 | date = August 2009 | pmid = 19620998 | doi = 10.1038/nchembio.203 }} or to detect the presence of certain molecules using proteins. For instance, the GFP-calmodulin fusion described above can be used to detect the level of calcium ions in a sample.

Many sequence alignment and protein structure alignment algorithms have been developed assuming linear data representations and as such are not able to detect circular permutations between proteins. Two examples of frequently used methods that have problems correctly aligning proteins related by circular permutation are dynamic programming and many hidden Markov models. As an alternative to these, a number of algorithms are built on top of non-linear approaches and are able to detect topology-independent similarities, or employ modifications allowing them to circumvent the limitations of dynamic programming. The table below is a collection of such methods.

The algorithms are classified according to the type of input they require. Sequence-based algorithms require only the sequence of two proteins in order to create an alignment. Sequence methods are generally fast and suitable for searching whole genomes for circularly permuted pairs of proteins. Structure-based methods require 3D structures of both proteins being considered.{{cite journal | vauthors = Prlic A, Bliven S, Rose PW, Bluhm WF, Bizon C, Godzik A, Bourne PE | title = Pre-calculated protein structure alignments at the RCSB PDB website | journal = Bioinformatics | volume = 26 | issue = 23 | pages = 2983–5 | date = December 2010 | pmid = 20937596 | pmc = 3003546 | doi = 10.1093/bioinformatics/btq572 }} They are often slower than sequence-based methods, but are able to detect circular permutations between distantly related proteins with low sequence similarity. Some structural methods are topology independent, meaning that they are also able to detect more complex rearrangements than circular permutation.

{{Academic peer reviewed|Q5121672|doi-access=free}}

{{reflist|colwidth=30em}}

• David Goodsell (April 2010) [http://www.rcsb.org/pdb/101/motm.do?momID=124 Concanavalin A and Circular Permutation] Protein Data Bank (PDB) Molecule of the Month

{{Good article}}

• {{PDBe-KB2|P02866|Concanavalin-A}}

Category:Proteins

Category:Permutations