The tetratricopeptide-repeat motif is a versatile platform that enables diverse modes of molecular recognition

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ScienceDirect The tetratricopeptide-repeat motif is a versatile platform that enables diverse modes of molecular recognition Albert Perez-Riba1 and Laura S Itzhaki2 Tetratricopeptide repeat (TPR) domains and TPR-like domains are widespread across nature. They are involved in varied cellular processes and have been traditionally associated with binding to short linear peptide motifs. However, examples of a much more diverse range of molecular recognition modes are increasing year by year. The Protein Data Bank has an ever-expanding collection of TPR proteins in complex with a myriad of different partners, ranging from short linear peptide motifs to large globular protein domains. In this review, we explore these varied binding modes. Additionally, we hope to highlight an emerging property of this simple, malleable fold—the potential for programmable complexity that can be achieved by acting as a scaffold for multiple binding partners. Addresses 1 Donnelly Centre for Cellular & Biomolecular Research, University of Toronto, Toronto, Canada 2 Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK Corresponding authors: Perez-Riba, Albert ([email protected]), Itzhaki, Laura S ([email protected])

Current Opinion in Structural Biology 2019, 54:43–49 This review comes from a themed issue on Folding and binding Edited by Ylva Ivarsson and Per Jemth

https://doi.org/10.1016/j.sbi.2018.12.004 0959-440X/ã 2018 Elsevier Ltd. All rights reserved.

Introduction Tandem-repeat domains are one of the most common classes of protein architectures (Figure 1). Their frequency is probably a result of replication slippage and recombination events on the DNA [1,2]. These mechanisms are considered sources of hypermutability, which has resulted in a high polymorphism compared to the background rate of point mutations [2–4]. The a-solenoid tandem-repeat proteins comprise repeats of a hairpin of antiparallel a-helices that are between 12 and 45 amino acids in size [2]. Armadillo repeats [5], HEAT (Huntingtin, elongation factor 3 (EF3), protein phosphatase 2A (PP2A) subunit, PI3-kinase TOR1) repeats [6,7] and www.sciencedirect.com

tetratricopeptide repeats (TPR) [8] are the most common members of this fold. There are clear structural differences between them; however, classification is determined more by sequence similarity rather than by structure [1,2,9–12]. In this review, we focus on the TPR proteins and, in particular, the diverse binding modes in which they engage. TPRs, as the name indicates, are 34-amino acid motifs that are found in arrays comprising between 2 and 20 repeats, wherein the repeats pack against each other giving rise to coiled and superhelical structures. The motifs fold in a helix-turn-helix conformation and are linked to the next repeat by a short loop, usually consisting of four residues. Over the past 20 years, there has been a steady increase in the number of TPR structures in the Protein Data Bank (PDB). Each new structure adds further detail to the picture of an exceptionally versatile fold capable of mediating protein–protein interactions via varied mechanisms.

Short peptide binding to the TPR concave groove This binding mode is most well represented in the PDB. Usually, a short peptide of around five amino acids binds to the concave groove formed by the TPR solenoid over a span of 2–3 repeats. The relatively large exposed surface area per residue provided by a short linear motif (SLiM) compared with a folded domain allows moderate affinity interactions to be encoded in just a few residues [13], and the binding affinities that have been measured for these TPR–peptide interactions to date are mostly in the low-micromolar to mid-micromolar range. The bound peptides can adopt an extended, kinked, turnlike or alpha-helical structure in the complex with the TPR protein [14]. There is a large variation of peptide sequences that interact with TPRs in this mode (Table 1.I), reflecting the variability of amino acid composition of the solventexposed interface on the TPRs [15]. Nevertheless, these complexes in the PDB are dominated by a very specific sequence, first identified in the C-terminal domain of Hsp90. This peptide is highly negatively charged (MEEVD) and must be located at the C-terminus, as the C-terminal carboxylate group makes key contacts with the TPR protein. The TPR residues involved in binding the MEEVD peptide are referred to as a ‘carboxylate clamp’ [8,16,17]. Since the first structure of the TPR domain of HOP (Hsp70–Hsp90 organising Current Opinion in Structural Biology 2019, 54:43–49

44 Folding and binding

Figure 1

(a)

(b)

(c)

(d)

(e)

(f)

(h)

(g)

Current Opinion in Structural Biology

Examples of TPR domains (cyan) bound to their peptide target (red). (a) TPR domain of HOP bound to an MEEVD peptide (PDB: 1ELR). (b) TPR domain of Fis1 bound to a helical segment of Mdv1 (PDB: 2PQN). (c) TPR domain of PEX5 bound to PST1. The hinge region of PEX5 located between the two TPR subdomains is highlighted in orange (PDB: 3CV0). (d) TPR domain of KLC1 bound to the Tyr acidic C-terminal peptide of torsinA (PDB: 6FV0). (e)–(g) LGN domain bound to NuMA, Inscuteable and Afadin, respectively (PDB: 3RO2, 3SF4, 5A6C). (h) TPR domain of CTR bound to Paf1 (red) and Cdc73 (orange) (PDB:6AF0).

Table 1 Binding mechanisms of TPR proteins in the PDB Binding mechanism

TPR protein (PDB filename)

I. Short peptide binding to the TPR concave groove

HOPa (1ELR), CHIPa (2C2L, 4KBQ), PP5 (2BUG)a, NrfG (2E2E), Tom71a (3FP2), CTPR390 (3KD7)a, Tah1a (2L6J, 4CGU, 4CGQ), SYCD (4AM9), UNC-45a (4I2W), PcrH (4JL0), ComR (5JUB), Sgt2a (5LYN), FKBP51a (5NJX), Fis1 (2PQN), Bub1 (4A1G), FKBP38a (5MGX), RPAP3a (6FDT, 6FDP), OM64a (6HPG), C-P4H (6EVM, 6EVN, 6EVO), KLC2 (3ZFW, 6FV0, 6FUZ)

II. Long extended peptide binding to the TPR concave groove PknG (2PZI), LGN (3RO2, 3SF4, 4A1S, 4G2V, 4WND, 5A6C), NprR (4GPK), OGT (4N39, 5LVV), Scc2 (5C6G), SRP68-72 (5M72), CTR9 (6AF0) III. TPR hinge formation

PEX5 (1FCH, 2C0L, 2J9Q, 3CV0, 3R9A), Trip8b-1a (4EQF)

IV. Binding of folded domains to the TPR protein

P67Phox (1E96), PP5 (1WAO), COP-e (3MKQ), BamD (3TGO), TatT (4DI3), NatA (4KVM), Ski2 (4BUJ), Anaphase promoting complex (APC/C) subunit (4UI9), TTC7B (5DSE), Hif1 (5BT1), HAP40 (6EZ8)

V. Homo-dimers, homo-oligomers and domain swap

CTPR3Y3 (2WQH), LapB (4ZLH), SART3 (5CTQ), Sgt1 (5AN3), TTC0263 (2PL2), MamA (3AS4), SNX21 (4YMR), KLC2 (6EJN)

a

Indicates binding to an MEEVD motif.

protein) bound to the Hsp90 peptide 18 years ago [18], many new TPR co-chaperones have since been crystallised, and their interactions with MEEVD peptides have been thoroughly characterised (e.g. yeast co-chaperone Current Opinion in Structural Biology 2019, 54:43–49

Sgt2 [19], human FKBP51 [20] and FKBP38 [21], and most recently RPAP3 [22]). Additionally, the Regan lab has made a series of specific TPR-cognate peptide pairs by varying the sequence of both components [23]. www.sciencedirect.com

Diverse binding modes of tetratricopeptide repeat proteins Perez-Riba and Itzhaki 45

Another recent discovery is a group of TPR proteins involved in the movement of cargo via microtubules. Kinesin proteins recognise their cargo through the kinesin light chain TPR domain (KLC1 and KLC2). Originally, a cargo recognition mechanism was identified involving a short linear motif in the cargo proteins known as the tryptophan acidic motif. This motif binds across the concave surface formed from three TPRs of kinesin light chain [24]. Recently, a second motif, the tyrosine acidic motif, was identified to bind preferentially to KLC1. Interestingly, the binding surfaces on the TPR domain for the two peptide motifs partly overlap, and this overlap is sufficient that the motifs compete with each other for binding to the TPR repeats and cannot both be bound at the same time [25].

Long extended peptide binding TPR concave groove In striking contrast to the short peptides most commonly associated with TPR binding, longer peptides binding to a more extensive surface area of the TPR are also observed (Figure 2d–g). The solenoid structure of Figure 2

(a)

(b)

A second example is the long 13-TPR domain of the Olinked b-N-acetyl-D-glucosamine Transferase (OGT). OGlcNAcylation is an important postransductional modification for the regulation of many cytoplasmic processes and has been linked to neurodegeneration [31]. The TPR domain was known to be involved in substrate recognition, but only recently has a mechanism been determined showing that mutation of an Asn ladder in the TPR domain greatly diminishes cellular O-GlcNAcylation [32]. An Asn ladder is formed in this TPR protein, similar to that in the LGN proteins formed by the Asn of the Leu-Gly-Asn residues [30].

(c)

Current Opinion in Structural Biology

Examples of TPR (or TPR-like) domains (cyan) in complex with their folded binding partners (red). (a) Structure of TTC7B TPR domain bound to FAM126A (PDB:5DSE). (b) Structure of the Hif1 TPR domain bound to the H2A–H2B heterodimer (PDB:5BT1). (c) CryoEM structure of HAP40, a TPR-like domain in complex with Huntingtin protein (PDB: 6EZ8). www.sciencedirect.com

the TPR scaffold generates a concave groove whose surface area increases with the number of repeats. Long peptides of 20–40 amino acids are known to interact across the length of the solenoid via the groove. The best-studied example is the TPR domain of the LeuGly-Asn rich (LGN) protein, an 8-TPR array with a characteristic LGN sequence at residues seven–nine of each TPR unit. The LGN protein was found to have crucial roles in symmetric and asymmetric cell division through binding to a number of unstructured peptides with high affinities in the mid-nanomolar range as a result of the large interface surface area [26]. The first proteins to be identified as binding to LGN were the Nuclear Mitotic Apparatus protein (NuMA) and Inscuteable [27,28,29], and more recently a third protein, Afadin, was found to bind to LGN in a similar manner. The Inscuteable peptide is almost 40 residues in length and can be roughly divided into three binding modules: an a-helix, an extended region, and an antiparallel b-sheet, the two b-strands or which are connected by a 13-residue loop. The Afadin and NuMa peptides interact in a similar, though not identical, way to Insucteable. Afadin is also capable of binding Factin, which provides a link between the LGN protein and actin in spindle organisation during cell division [30]. Ultimately, this is a nice example of how the same binding groove of the TPR proteins can accommodate different partners with orders of magnitude differences in affinity that may allow for fine-tuned regulation of multiple cellular processes.

Recently, another striking example has come to light. The Paf1 complex is a general RNA polymerase II transcription elongation factor in eukaryotes. The core of the complex is formed by CTR9, a 21-repeats TPR domain, and the Paf1 protein. The 120-residue intrinsically disordered N-terminal domain of Paf1 binds the full span of the CTR9 solenoid across its vertical axis (Figure 1h). This forms a tight core where other members of the complex assemble on top. A third member was cocrystalised. A 73-residue peptide from Cdc73 comprising three alpha-helices that wrap around one of the turns of the solenoid. Interestingly, part of Cdc73 binds to the concave groove of CTR9 already occupied by Paf1. Current Opinion in Structural Biology 2019, 54:43–49

46 Folding and binding

Bound Paf1 presumably generates a new interface necessary for Cdc73 binding.

hinge formed by TPR4 allows for the formation of a cavity that encapsulates the PST-1 peptide [39–41].

In summary, this binding mode of the TPR proteins resembles that used by the armadillo-repeat proteins (such as beta-catenin [33,34]), and it has recently been exploited by Plu¨ckthun and co-workers to make artificial armadillo-repeat proteins designed to recognise specific peptide sequences [35,36]. TPR domains are generally considered to be rather rigid binding scaffolds that respond to ligand binding with limited conformational changes. However, comparisons between the structures of the TPR domains in their uncomplexed states and those in complex with the very long peptides described in this section reveal dramatic conformational differences, whereby peptide binding induces an ‘open-to-closed’ transition in the solenoid structure, and suggestive of an induced-fit mechanism.

Binding of folded domains to the TPR protein

TPR hinge formation The relatively small interfaces produced by the binding of a short peptide motif to the TPR groove prevent highaffinity interactions. This is not necessarily bad: living systems need weak transient interaction to function. However, it poses a problem if we want to optimise the affinity of a TPR–peptide interaction. Cortajarena et al. were able to obtain libraries of peptide-binding TPR proteins based on HOP [23], but the affinities do not match other binding scaffolds such as the above-mentioned armadillo repeats or ankyrin repeats or classic antibodies and antibody alternatives [36,37]. One might imagine that evolution would encounter the same problem. A solution to this problem may be to use an interface along the solenoid axis rather than perpendicular so as to increase the surface area and the peptide sequence space that can be explored. Another solution can be found in the interaction between the PEX5 receptor and the peroxisomal-targeting sequence-1 (PTS-1). Like the LGN protein, PEX5 is an 8-TPR array. However, unlike the long LGN-binding peptides, the PEX5-binding peptide from PTS-1 is short (5 residues). Nevertheless, the affinities reported for PTS-1 binding to PESX5 are in the low-to-mid nanomolar range [38]. Thus, there has been an increase in buried surface area, but clearly not through an increase in the peptide length and thereby the interface area. The fourth repeat of the PEX5 TPR domain does not have a typical TPR sequence and does not adopt the TPR fold, instead forming a ‘hinge’. Thus, in contrast to the straight, continuous solenoid formed by the 8-TPR array of LGN, the TPR domain of PEX5 is splits into two separate subdomains linked by the hinge. The 5-residue peptide of PTS-1 binds across TPR3, TPR4, and TPR5 (i.e. three repeats, as for the interaction between Hop and Hsp90). However, additionally in this case, the Current Opinion in Structural Biology 2019, 54:43–49

As seen in Table 1, the structural database of TPR complexes is heavily dominated by short peptide interactions. The most well studied of those being hsp90 with dozens of PDB entries. Nevertheless, TPR proteins are capable of binding folded domains across different sections of the solenoid structure. This ability elevates TPR into a higher level of complexity, making them crucial parts of large protein complexes, or key players in the regulatory mechanism. A classic example is the TPR domain of PP5, which self-inhibits the phosphatase activity through extensive contacts across three of the repeats (turn region) and the globular catalytic domain of the enzyme [42]. The domain is inhibited simply by the occlusion of its active site. On the other side, Hsp90 regulates the activity of PP5 through binding to the TPR domain, which conserves its carboxylate clamp motif at the concave groove. In turn, this interaction competes against the catalytic domain of PP5 that partially blocks the carboxylate clamp with its helical Cterminus [42]. Another well studied system is P67Phox, which contains a 4-repeat TPR array with the solvating helix. This TPR domain displays a structured loop connecting repeats TPR3 and TPR4 and a long extended Cterminal tail that folds back and binds to the TPR domain itself, establishing contacts with the structured loop and stabilizing its b-turn structure. The TPR domain of P67Phox then binds the GTPase protein Rac through the surface created by the structured loop, a crucial step in the formation of the NADPH oxidase complex [43]. A more recent example is the phosphatidylinositol 4kinase complex, a multi-protein membrane complex responsible for the production of phosphatidylinositol 4-phosphate. The complex is formed by a membrane anchoring domain (EFR3B), a regulatory domain, (FAM126A), a catalytic domain (PI4KIIIa), and a 14repeat TPR superhelix (TTC7) that coordinates the multiprotein assembly. The binding mechanism of TTC7 and PI4KIIIa or EFR3B is not well understood, but TTC binds the folded FAM126A though the large surface area created by the groove of the superhelix formed by eight repeats. Biochemical data suggest that both PI4KIIIa and EFR3B must also bind at different regions of TTC7 to achieve the fully functional complex attached to the membrane (Figure 2a) [44]. Another recent example of such capabilities is seen in the recent structure of Huntingtin (HTT). The structure of this important protein was finally resolved though cryoEM thanks to the stabilising interaction with the large TPR-like protein HAP40. HTT is by itself a protein made of a-solenoid HEAT-repeat domains. Two of these HEAT domains are separated by a bridge domain consisting predominantly of a-helical (non-HEAT www.sciencedirect.com

Diverse binding modes of tetratricopeptide repeat proteins Perez-Riba and Itzhaki 47

unassigned) repeats. HTT by itself has too much conformational heterogeneity to be resolved by EM, a problem solved by its stabilisation upon binding to HAP40. HAP40 comprises seven tetratricopeptide-like repeats and appears to be unfolded until it binds to HTT. HAP40 binds between the two HEAT domains of HTT [45]. This example shows the potential of TPRs and other solenoids in arranging themselves in large complexes, as first shown by the cryoEM structures of TPR subunits of the anaphase-promoting complex (APC) [46]; additionally, it illustrates the problems when classifying TPRs and other similar alpha-solenoids [47]. The HAP40 repeats have been classified by the authors as TPR-like because sequence identity does not classify them as TPR repeats, although structurally they show high homology to the TPRs. The final recent discovery we present is Hif1, a histone chaperone, whose TPR domain binds the H2A–H2B dimer and the H3–H4 tetramer through two different interfaces of the TPR protein. Again, multiple contacts across the Hif1 TPR solenoid with the globular domains of the histone dimers are required for the interaction. Interestingly, binding to the two different histones (H2A– H2B and H3–H4) is mutually exclusive. Although there are no direct steric clashes that would prevent both histone complexes from binding to Hif1 simultaneously, the authors suggest that the binding of one histone complex would cause a change in the binding interface for the other [48]. Allostery in TPRs is a fascinating property that is well described in the RRPNN subclass of TPRs which we discuss in detail in our recent opinion piece [49]. A further property of TPRs, though outside the scope of this review, is their ability to form homo-dimers and homo-oligomers, which has been exploited by both nature and researchers as the basis for assembly scaffolds and biomaterials [50,51,52]. A collection of interesting TPR oligomer structures can be found in Table 1.

Summary Here, our aim is not simply to list the most recently identified TPR proteins or the many TPR binding modes observed to date. Instead, we hope to bring together examples that illustrate the extraordinary scope of this protein fold and hint at how many of its capabilities have yet to be exploited. TPR proteins can recognise both short peptides and large globular domains, with binding affinities ranging over six orders of magnitude. TPR domains are commonly found within multi-domain enzyme and often play crucial roles in regulating the activity of the catalytic domain for example by modulating substrate specificity [16,29,53]. Repeats can be continuously stacked one upon the other, allowing for the inline addition of new capabilities without affecting the existing one [44,48]. This property enables, for example, www.sciencedirect.com

a TPR domain to bind to its catalytic partner and induce enzyme inactivation while still being able to bind to a peptide and prevent inhibition, leading to an additional layer of regulation [42]. Ultimately, TPRs can then be key parts of large complexes and scaffolds for the assembly and organisation of multiple partners on top of them [44,46]. It is this facility to stack simple individual functions (such as binding to a peptide), one after the other or in combination with each other, into larger elements with complex programmed functions that constitute the really great promise of TPRs for applications in medicine and biotechnology. Moreover, some TPRs have been shown to be extremely thermostable [54,55], whereas others mostly unstructured and only capable of folding upon binding to their partners [45]. Information can be transmitted through them in the form of allosteric effects, as seen by the mutually exclusive binding to H2A–H2B or H3–H4 histones [48]. These features, thereby, allow TPR proteins to act as molecular switches transforming chemical information, such as a peptide, into a metabolic cascade [49]. Yet further TPR capabilities, such as binding to RNA [56] and TPR homo-dimer/oligomer formation, are discussed elsewhere [57,58]. In all, the molecular recognition information of TPR proteins is encompassed in sequential repetitions of a small motif with as few as eight highly conserved residues at the core positions [15]. In a way, the true application of TPR proteins in translational science may not be as highaffinity target binders. This is a task that designed ankyrin repeats [59] and armadillo repeats [36] have already been shown to accomplish very effectively. TPRs appear to us to be much better suited to be bi-functional and multifunctional molecules, where complex interactions between molecules are programmed in line.

Acknowledgements Research in the Itzhaki lab is funded by the UK Biotechnology and Biological Sciences Research Council and Cancer Research UK.

Declaration of Interest statement Authors AP and LSI are inventors on patent application PCT/EP2018/ 068580Authors AP and LSI are founders of PolyProx Therapeutics Limited (company number 11664980).

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Diverse binding modes of tetratricopeptide repeat proteins Perez-Riba and Itzhaki 49

and rigid domain motions in ligand binding. BMC Struct Biol 2007, 7:24. 41. Sampathkumar P, Roach C, Michels PAM, Hol WGJ: Structural insights into the recognition of peroxisomal targeting signal 1 by Trypanosoma brucei peroxin 5. J Mol Biol 2008, 381:867-880. 42. Yang J, Roe SM, Cliff MJ, Williams MA, Ladbury JE, Cohen PTW, Barford D: Molecular basis for TPR domain-mediated regulation of protein phosphatase 5. EMBO J 2005, 24:1-10. 43. Lapouge K, Smith SJ, Walker PA, Gamblin SJ, Smerdon SJ, Rittinger K: Structure of the TPR domain of p67phox in complex with Rac.GTP. Mol Cell 2000, 6:899-907. 44. Baskin JM, Wu X, Christiano R, Oh MS, Schauder CM, Gazzerro E, Messa M, Baldassari S, Assereto S, Biancheri R et al.: The leukodystrophy protein FAM126A (hyccin) regulates PtdIns(4) P synthesis at the plasma membrane. Nat Cell Biol 2016, 18:132-138. 45. Guo Q, Bin Huang B, Cheng J, Seefelder M, Engler T, Pfeifer G,  Oeckl P, Otto M, Moser F, Maurer M et al.: The cryo-electron microscopy structure of huntingtin. Nature 2018, 555:117-120. Gouet al. were ultimately able to obtain a 3D structure of the Huntingtin protein thanks to complexing to a TPR-like domain, HAP40. Huntingtin is mostly unfolded in solution, whereas Hap40 appears to be intrinsically unfolded. Nevertheless, a large folded complex is formed upon binding, and thus it could be resolved through electron microscopy. The structure of Huntingtin will be of obvious importance in the research for new drugs to tackle Huntington disease. 46. Chang L, Zhang Z, Yang J, McLaughlin SH, Barford D: Atomic structure of the APC/C and its mechanism of protein ubiquitination. Nature 2015, 522:450-454. 47. Di Domenico T, Potenza E, Walsh I, Parra RG, Giollo M, Minervini G, Piovesan D, Ihsan A, Ferrari C, Kajava AV et al.: RepeatsDB: a database of tandem repeat protein structures. Nucleic Acids Res 2014, 42:D352-D357. 48. Zhang M, Liu H, Gao Y, Zhu Z, Chen Z, Zheng P, Xue L, Li J,  Teng M, Niu L: Structural insights into the association of Hif1 with histones H2A-H2B dimer and H3-H4 tetramer. Structure 2016, 24:1810-1820. Hif1 is a TPR protein involved in regulation of histone function. The authors found Hif1 to bind both H2A–H2B and H3–H4 histone complexes in a mutually exclusive manner. Steric factors are not the underlying reason. Therefore, some allosteric effect upon binding must occur to regulate binding to the complexes. This study brings to light a new layer of regulation in histone function and chromatin regulation. 49. Perez-Riba A, Synakewicz M, Itzhaki LS: Folding cooperativity  and allosteric function in the tandem-repeat protein class. Philos Trans R Soc B Biol Sci 2018, 373 20170188. Allostery is a well-studied property in enzymatic reactions but is not understood for what is generally believed to be rigid scaffolds involved in

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protein assembly. However, it is now becoming apparent that the intrinsic physical properties that define tandem-repeat protein folding also allow for a flexible, breathing scaffold through which information can be transferred. In this opinion piece, the authors summarise the latest findings in the RRPNN class of TPR proteins and hypothesise the mechanisms that may be at play in the allostery of repeat proteins. 50. Sanchez-deAlcazar D, Mejias SH, Erazo K, Sot B, Cortajarena AL:  Self-assembly of repeat proteins: concepts and design of new interfaces. J Struct Biol 2018, 201:118-129. Sanchez-deAlcazaret al. explored three different design strategies to generate new interfaces across the solenoid groove of a consensus TPR protein to promote homodimerisation. On the basis of the known structure of the CTPR superhelix, the authors expected the formation of a compact structure with a pore all the way through. Impressively, all their three strategies generated homodimers with the expected geometry. This is a wonderful showcase of the tremendous malleability of the TPR motif to design new tools and biomaterials. 51. Mejias SH, Aires A, Couleaud P, Cortajarena AL: Designed repeat proteins as building blocks for nanofabrication. Advances in Experimental Medicine and Biology. 2016:61-81. 52. Grove TZ, Cortajarena AL: Protein design for nanostructural engineering: concluding remarks and future directions. Advances in Experimental Medicine and Biology. 2016:281-284. 53. Deng P, Zhou Y, Jiang J, Li H, Tian W, Cao Y, Qin Y, Kim J, Roeder RG, Patel DJ et al.: Transcriptional elongation factor Paf1 core complex adopts a spirally wrapped solenoidal topology. Proc Natl Acad Sci U S A 2018, 115:9998-10003. 54. Phillips JJ, Javadi Y, Millership C, Main ERG: Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors. Protein Sci 2012, 21:327-338. 55. Cortajarena AL, Lois G, Sherman E, O’Hern CS, Regan L, Haran G: Non-random-coil behavior as a consequence of extensive PPII structure in the denatured state. J Mol Biol 2008, 382:203-212. 56. Johnson B, VanBlargan LA, Xu W, White JP, Shan C, Shi P-Y, Zhang R, Adhikari J, Gross ML, Leung DW et al.: Human IFIT3 modulates IFIT1 RNA binding specificity and protein stability. Immunity 2018, 48:487-499.e5. 57. Lim H, Kim K, Han D, Oh J, Kim Y: Crystal structure of TTC0263, a thermophilic TPR protein from Thermus thermophilus HB27. Mol Cells 2007, 24:27-36. 58. Zeytuni N, Ozyamak E, Ben-Harush K, Davidov G, Levin M, Gat Y, Moyal T, Brik A, Komeili A, Zarivach R: Self-recognition mechanism of MamA, a magnetosome-associated TPRcontaining protein, promotes complex assembly. Proc Natl Acad Sci U S A 2011, 108:E480-E487. 59. Tamaskovic R, Simon M, Stefan N, Schwill M, Plu¨ckthun A: Designed ankyrin repeat proteins (DARPins) from research to therapy. Methods Enzymol 2012, 503:101-134.

Current Opinion in Structural Biology 2019, 54:43–49