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Metamorphic Proteins: Emergence of dual protein folds from one primary sequence Muralikrishna Lella, and Radhakrishnan Mahalakshmi Biochemistry, Just Accepted Manuscript • Publication Date (Web): 01 Jun 2017 Downloaded from http://pubs.acs.org on June 5, 2017

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Metamorphic Proteins: Emergence of dual protein folds from one primary sequence Muralikrishna Lella1,* and Radhakrishnan Mahalakshmi1,* 1

Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of

Science Education and Research, Bhopal – 462066. India. *E-mail: [email protected]; [email protected].

KEYWORDS. Metamorphic proteins, fold switching, α-helix, β-sheet, chameleonic sequences.

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ABSTRACT

Every amino acid exhibits a different propensity towards distinct structural conformations. Hence, decoding how the primary amino acid sequence transitions to a defined secondary structure and its final three-dimensional fold is presently considered predictable with reasonable certainty. However, protein sequences have recently been discovered that defy the first principles of secondary structure prediction – they attain two different folds. Such proteins, aptly named metamorphic proteins, decrease the conformational constraint by increasing flexibility in the secondary structure, and thereby result in efficient functionality. In this review, we discuss the major factors driving conformational switch, relating both to protein sequence and structure using illustrative examples. We discuss the concept of evolutionary transition in sequence and structure, the functional impact of the tertiary fold, and the pressure of intrinsic and external factors that give rise to metamorphic proteins. We mainly focus on the major components of protein architecture, namely, the α-helix and β-sheet segments, which are involved in conformational switching within the same or highly similar sequences. These chameleonic sequences are widespread in both cytosolic and membrane proteins, and these folds are equally important for protein structure and function. We discuss the implications of metamorphic proteins and chameleonic peptide sequences in de novo peptide design.


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INTRODUCTION Predicting the structure of newly identified or designed protein sequences based on homology modeling using computational algorithms has seen considerable advances in the past four decades. The accuracy of prediction has improved to 80%;1-3 however, the complexity in protein folds severely hampers complete de novo structure prediction. Several recent examples of proteins and de novo designed peptide sequences have been reported, which defy Anfinsen’s ‘one sequence, one fold’ paradigm to varying degree.4 The observation of such “chameleonic sequences” that exhibit structural plasticity and toggle between dual folds renders structure prediction a challenging task. The available protein structures in the PDB (Protein Data Bank) are increasing with time, emphasizing the need to revisit protein structures that exhibit structural plasticity. Here, we discuss recently identified examples of protein and peptide sequences adopting multiple structures, and we discuss the molecular basis of such changes. The major focus on chameleonic sequences started in 1980s when work from Kabsch and Sander (1984),5 and the Brenner group (1985)6 reported that short sequences from different proteins could attain two distinct folds. Analysis of the structures of 62 proteins revealed distinct structural variability even in short sequences that were five residues long.5 Subsequently, Cohen’s group extended the analysis to identical hexapeptide sequences from a larger dataset of 316 proteins.7 They showed that identical sequences can exhibit vastly different structures, and can exist in unrelated classes of protein folds. For example, Cohen’s group identified eight classes of sequences that exhibited α-helical structure in one class and formed β-strands in another class of proteins.7 In 1996, Minor and Kim coined the term ‘chameleon sequences’ for such sequences, when they demonstrated the context-dependent structural conversion of a 11-


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residue segment of a protein.8 Subsequently, Mihaly Mezei used a larger PDB structural dataset to show that the longest naturally occurring chameleonic sequences are seven residues in length.9 Several other examples of chameleonic sequences (heptapeptide and octapeptide) have since been observed.10, 11 More recently, attempts to improve protein structure prediction efficiency, and identification of evolutionary relationship between secondary structures with the same amino acid sequence, have led to the identification of conformational flexibility and helix-strand transitions in short peptides from unrelated proteins.12, 13 Subsequently, several studies have identified molecular and environmental factors that drive conformational conversion in chameleonic sequences.14-16 Studies have deduced the mechanism of structural interconversion experimentally and through molecular dynamics simulations (MDS) in both chameleonic sequences in general, as well as amyloidogenic proteins in particular.17-21 The well-established phenomenon of amyloid formation involves the structural transition from α-helix to β-sheet; the latter then assembles into ordered β-aggregates. This conformational interconversion observed during amyloidogenesis has been investigated and discussed extensively in the literature. Hence, this review does not include examples of β-aggregates. This review mainly focuses on conformational transitions observed for non-amyloidogenic sequences in different circumstances.

ENERGY BARRIER AND SEQUENCE SPACE The protein universe is largely made up of 20 amino acids in its primary sequence. Permutations in the secondary structure elements of α-helix, β-sheet, and random coil bring additional diversity in protein structure and facilitate its function. The diversity we find in protein sequences and structures raises interesting questions such as: (i) What drives the primary


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amino acid sequence to reach a defined secondary structure? (ii) How much freedom of fold space is available to the protein in its environment? (iii) How do the fold and sequence dictate protein function? It is pertinent to now address these questions in the context of structural plasticity exhibited in chameleonic sequences. Evidently, factors controlling protein sequencestructure-space serve as parameters that are together responsible for the ultimate fold that the protein adopts.22, 23 Flexibility in local interactions mediated through sequence-space readjustment could allow the folded protein to escape a unique energy minimum and populate a second favorable energy in the folding pathway.20 The emergence of chameleonic sequences therefore relies on an evolutionarily optimized energetic equilibrium between at least two unrelated structures.24-26 It is well known that, folded proteins exist as an ensemble of closely related equilibrium structures in their energetically stable state. However, the conundrum in metamorphic proteins is that more than one distinct structural form is energetically favored. An evolutionary optimization of a single sequence between two equally stable structures indicates that structural (scaffold) plasticity is thermodynamically allowed.27 Further, oligomerization of one of the two metamorphic proteins is believed to energetically stabilize the fold, and facilitate the emergence of alternate structural scaffolds.28 Changes in environmental factors such as buffers or lipidic conditions can also energetically favor the bidirectional equilibrium of a single sequence towards one scaffold or structural assembly.29 This is achieved by changes to the energy landscape such that one structural form is more stabilized than another. An accompanying high energy barrier for the reverse process “locks” the protein in one metamorphic state.29 In proteins wherein oligomerization of at least one of the metamorphs is not mandated by the free energy landscape, the primary sequence is believed to possess stabilizing and destabilizing


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segments. Structural rearrangement usually requires a near-complete unfolding of one of the forms and re-establishment of new non-covalent interactions in the alternate metamorphic form.30 An exhaustive study of human protein sequences by the Hilser group points to the existence of a thermodynamic environment31 that balances structure and sequence compatibility. The authors demonstrate that negative selection pressure leads to the retention of one (or a subset) of folds in a system where energetic compatibility of one sequence for more than one fold can exist. This negative selection is imposed by incompatible sequences that function as gatekeeper sequences in deciding folds that are disallowed. Hence, evolution seems to function in contrast to the positive design (or positive compatibility index) that is usually employed in de novo design and protein engineering,32 wherein favorable interactions are preferably retained. The benefits of negative design have also been demonstrated in other studies.33 It can be envisioned that metamorphic proteins have retained some gatekeeper residues31 in sequence and structure segments that determine conformational specificity. Yet, it is likely that they also possess residues of low negative compatibility (high positive compatibility index) that promote alternative favorable interactions and thermodynamically support the formation of more than one stable fold. The following sections now discuss how intrinsic and extrinsic factors allow the folding intermediate to choose between two distinct folded protein states, with illustrations drawn from structural and functional proteins.


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HISTORY OF THE PARACELSUS CHALLENGE In 1994, in an attempt to understand protein folding, Creamer and Rose challenged the scientific community to achieve two different folds from one protein by changing no more than 50% of the protein sequence.34, 35 They termed this “The Paracelsus Challenge” for the protein world. Several groups solved this problem to varying degree, but the results from three different groups – Paracelsin-43 from the Thoronton group in 1996, Crotein-G from Yuan and Clarke in 1998, and Janus from the Regan group in 1997 – merit discussion (Figure 1). All three amended proteins involved the redesign of the parent protein cores.36

Figure 1: Reply to the Paracelsus challenge. In each panel, the parent protein is shown on the left, and the target protein is on the right. The residues retained from the parent and target proteins to attain the engineered proteins are highlighted in red and cyan color, respectively. The engineered proteins are Paracelsin-G from BDS-1 and B-domain of Protein A (panel A), CroteinG from 434 Cro repressor and B-domain of protein G (panel B), and Janus from B-domain of protein G and Rop protein (panel C). All the Protein Data Bank (PDB) entries are provided above each structure. The figures are reproduced and modified with permission from Dalal et al., 1997.36 Copyright © 1997 Elsevier Inc.


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a) Paracelsin-43: The Thornton group, in collaboration with the Sadler group, used the inverse protein folding method37, 38 to address the Paracelsus challenge. They chose two 90%.61-64 The domains GA98 and GB98 differed only at the 45th position, and possessed Leu and Tyr, respectively. The substitutions L45Y or Y45L in the GA98/GB98 system led to a change in the fold from 3α ↔ 4β+α.64, 65 The substitution at the 25th position from Thr to Ile in GB98 could also change the 4β+α fold to the 3α fold. This GB98-T25I (3α) structure could be rescued back to its 4β+α by substituting Leu to Ala at the 20th position (GB98-T25I, L20A).56, 66 The findings using protein G suggests that the conformational trigger in proteins can be confined to one or two amino acids.67 Such residues play a critical role in stabilizing tertiary interactions and do not allow any other structural rearrangement to occur. Highly homologous heteromorphic proteins such as protein G can pose substantial hurdles for computational biologists in predicting the secondary and three dimensional structure of a protein from the primary sequence. Yet, the identification of protein domains such as that seen for protein G can also provide interesting insight on fold switching and protein plasticity.


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CONFORMATIONAL CHANGE UPON PROTEIN DIMERIZATION a) Lymphotactin (Ltn10-Ltn40) Lymphotactin (Ltn, XCL1) is a protein from the C family of chemokines, and is involved in inducing chemotaxis in humans.68 Under physiological conditions (37 °C and 150 mM NaCl) Ltn exists as a dynamic equilibrium between two different native folds (monomer and dimer).69 Monomeric Ltn (Ltn10) presents a 3β+α form (Figure 6A), and binds specifically to its receptor XCR1 (GPCR family) present on leukocytes. Dimeric form of Ltn (Ltn40) is present in an all-β form (Figure 6B), and can bind to cell surface glycosaminoglycans,70 particularly heparin. Ltn40 does not activate XCR1; similarly, Ltn10 fails to recognize heparin. Hence, both structural states have specific affinity to their respective binding partners, and are therefore functionally isolated. The shift in population from Ltn10 to Ltn40 can be controlled under physiological conditions by a change in the salt concentration – Ltn10 is seen at high salt and low temperatures, while at higher temperatures of up to 40 °C and low ionic strength, Ltn40 is predominant.69 Ltn dimerization leads to a change in the protein hydrophobic core and long range interactions, which is achieved by altering the C-terminal α-helix to a β-sheet. Further, hydrogen bonds are reestablished with different amino acids.71


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Figure 6: Dynamic equilibrium between monomer and dimer with two different folds in Ltn/XCL1. (A) Ltn10 present in monomeric form adopts a 3β+α in high salt concentration and low temperature. (B) Ltn40 is present in the dimeric form with all-β conformation in low salt and higher temperatures. CC3 (C) and CC5 (D) are engineered Ltn proteins, designed by introducing cysteines (yellow) to lock the protein conformation either in monomer (C) or dimer (D) form through disulfide bonds. The region that changes from α-helix in Ltn10 to random coil region (in Ltn40) is shown as orange; the region that changes from random coil to β-sheet has been shown in blue. The color code for the proteins is retained in the multiple sequence alignment, except for cysteine which is shown as yellow/black. The PDB IDs are shown above each structure. Panel (A) and (B) are redrawn with inspiration from Tuinstra et al., 2008.71 Copyright 2008 National Academy of Sciences.


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In addition to identifying and characterizing the structural switch in Ltn, the Volkman group was able to show that Ltn could be conformationally locked in one of the two structures through disulfide bonds72, 73 and disturbing the hydrophobic core (W55→D).71 Disulfide bonds formed in the Ltn CC1 (T10→C and “AC” introduced between G32 and S33) and Ltn CC3 (V21→C, V59→C) variants locks the protein conformation in a monomeric form similar to Ltn10.72 The CC3 mutant shows characteristics similar to Ltn10, and can activate the XCL1 receptor (Figure 6C). In the Ltn CC5 (A36→C, A49→C) variant with a disulfide bond (Figure 6D), or Ltn W55→D with an altered hydrophobic core, the proteins are more stable in the dimeric form with high binding affinity to heparin.73 The stable dimers can also act as a potential HIV inhibitor.73 The Ltn10-Ltn40 equilibrium shows that a single protein can indeed perform multiple functions in the cell through structural transitions, and forms an outstanding example of a perfect metamorphic protein. Another example that shows a similar behavior is the conformational dimer of Mad2 with O-Mad2 and C-Mad2;74, 75 these systems have been reviewed elsewhere in greater detail.76, 77 b) Chloride Intracellular Channel Proteins (CLICs) The chloride intracellular ion channel proteins (CLICs)78, 79 are a family of intracellular anion channels that exhibit multiple metamorphic behavior – (i) they are present as soluble forms or transmembrane ion channels; (ii) one CLIC domain also undergo a conformational interconversion between β-sheet and α-helical structure, which decides whether the protein stays monomeric or forms a dimer.80 The N-terminal domain adopts an α3+β4 structure in its reduced form, which retains the protein as a monomer (Figure 7A). Oxidation modifies the cysteine residues, and leads to the formation of a disulfide bond between two conserved cysteines at the 24th and 59th positions.81 This oxidation causes the N-terminal domain to undergo a structural


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transition to an all-α state, which now dimerizes (Figure 7B). The intermediate dimeric state of CLIC inserts into the intracellular membrane and oligomerizes to form an anion channel. By studying the channel properties in the presence of reducing agents, and by using point mutations of the human CLIC protein CLIC1 (C24A), it was established that CLIC1 forms a redoxregulated functional channel.81

Figure 7: Redox-controlled dimerization of CLIC showing the conformational switch in the Nterminal domain. (A) The monomeric state of N-terminal CLIC has a 3α+4β form. Upon oxidation, cysteines present at the 24th and 59th positions (yellow) form a disulfide linkage, leading to protein dimerization (B). The dimeric interface can insert into the membrane. In both the structure and the schematic below, the change from a β-sheet structure to α-helix and loop regions has been shown in blue, and the change from unstructured region to α-helix has been shown in orange. The PDB IDs are shown above each structure.


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CONFORMATIONAL CHANGE UPON PROTEIN OLIGOMERIZATION One of the best examples of protein-protein interactions is oligomerization, wherein, homo- or hetero-oligomerization can occur. A stable and energy minimized oligomeric state can be achieved through several changes like domain repositioning, structural rearrangements, formation of new binding partners, and establishing inter- or intra-domain interactions. It is argued that oligomerization can promote conformational changes, by establishing new stabilizing interactions that are not seen in the monomeric protein.27 Both cytosolic and membrane proteins are known to form oligomerization-mediated metamorphic structures. Here, we discuss a few striking examples.

a) Cytosolic Proteins: Metallopeptidase A class of metallopeptidases – the selective and specific caseinolytic metallopeptidase (selecase) from Methanocaldococcus jannaschii – attracted recent attention for its highly metamorphic nature.82 Selecase is a highly soluble protein enzyme that exhibits a concentration– dependent oligomeric state, ranging from monomer (slc1) to octamer (slc8). The functional fold of the protein is in the monomeric state (Figure 8A). Selecase is inactive and self-inhibiting in the oligomeric states (dimer, tetramer, and octamer). The functionally active monomer contains two domains, namely the N-terminal subdomain (NTS, α/β sandwich) and C-terminal subdomain (CTS, all-α). A central hydrophobic core stabilizes the metal binding cleft; the amino acids responsible for catalysis are present between NTS α2 and the connecting loop to CTS α3. This region constitutes the NTS-CTS interface. Upon oligomerization, the stable tetrameric (slc4) form loses enzymatic activity by an auto-inhibitory structural rearrangement that occurs at the


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metal binding cleft (Figure 8B). In slc4, a connecting loop between helix α2-α3 and helix α3 protrudes away from the core of the protein and forms canonical anti-parallel β-strands β4 and β5. This conformational change displaces the metal binding ligand H80 by ~16Å and α4 by ~30Å in slc4, rendering selecase inactive. It has been observed that this conformational interconversion is completely concentration dependent, and repacking of monomeric or oligomeric states occurs spontaneously.82

Figure 8: Concentration-dependent oligomerization and structural transition of selecase. (A) The topology of helix α3 and connecting loop of α2-α3 (orange) in the monomeric form of selecase is highlighted. This region is involved in a conformation switch when the protein concentration increases. (B) Tetrameric state of selecase showing that the highlighted region adopts a β-hairpin structure that protrudes away from protein core. The PDB IDs are shown above each structure.


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b) Membrane Proteins: α- and β-Pore Forming Toxins Transmembrane proteins show limited variation in the structural motifs that also support their stability and function – they are predominantly α-helical in nature, while some outer membrane proteins of bacteria, mitochondria and chloroplast are β-sheet rich. The most interesting structural metamorphosis is exhibited by pore forming proteins.83 The pore forming proteins are a class of toxins produced from pathogens to attack the host cell by forming holes in the membrane. Pore forming toxins (PFT) are classified into two major classes, α-PFT and β-PFT, based on the final structural fold that is presented in the membrane spanning region.84, 85 PFTs exhibit high coordination between the protein segments as they undergo structural transitions from the monomeric soluble state to the oligomeric membrane integrated pore state. Cytolysin A (ClyA), also called hemolysin E (HlyE) from Staphylococcus aureus is categorized under α-PFT, and it forms α-helical channels in the membrane.86, 87 The structure of ClyA is a four α-helical bundle with one hydrophobic β-tongue (β-hairpin) that is stabilized by hydrophobic aromatic clusters present between the loop regions of two short α-helices αD and αE.86 The β-tongue undergoes a major conformational rearrangement upon ClyA assembly into the membrane, and converts into an α-helix. ClyA now forms three helical bundles with a membrane embedded helix αA1. The helical bundle associates in the membrane, forming a dodecameric transmembrane pore.86 The β-PFT pore formation mechanism has been studied extensively in both prokaryotes and eukaryotes. From the monomeric soluble form, the β-sheet or α-helical domain of the protein converts to an extended β-sheet and forms an oligomeric β-barrel pore in its membrane embedded form. Cholesterol-dependent cytolysin88 and the membrane attack complex/ perforin


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family89 show such kind of conformational transitions during the transmembrane pore formation. Other well-studied examples include the bacterial toxin pneumolysin,90 pleurotolysin complex (PlyA-PlyB, Figure 9),91 hemolytic lectin CEL-III,92 and lymphocyte perforin.93

Figure 9: Pore-forming toxins undergo structural transitions upon inserting into the membrane. Illustrated here is the example of the β-PFT class of proteins. PlyB of the PlyA-PlyB complex is involved in a conformational switch from α-helix to β-sheet (A-D, yellow color). This facilitates the formation of a pore in the membrane with a diameter of 80Å. The cross-section (E) and surface view (F) of the membrane pore are also provided. Figures are reproduced here with permission from Lukoyanova et al., 2015.91


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LIGHT-MEDIATED CONFORMATIONAL SIGNAL IN PHYTOCHROMES Light-mediated growth and development of cyanobacteria and plants require photoreceptors to harness light, and for efficient photosynthesis.94 One protein class involved in photomorphogenesis is the phytochrome (Phy) photoreceptor. Phytochrome shows photointerconversion between dark adopted red-light absorbing Pr state and the light activated far-red light absorbing Pfr state.95 Phy protein is divided into multiple domains.96, 97 The N-terminal photosensory module (PSM) bears the light absorbing segment. The output module, which stabilizes the head-to-head dimerization along its helical spine, is linked to the PSM. In addition, phytochromes contain the cGMP phosphodiesterase/ adenylyl cyclase/ FhlA (GAF) domain and the Phy-specific (PHY) domain. The GAF domain binds bilin while the PHY domain interacts with the photoreceptor bilin present in GAF domain and stabilizes the photo-activated state of the protein. PHY domain achieves this stabilization through a unique hairpin or tongue loop that extends outside the domain. The extended tongue loop contains two antiparallel β-sheets that interact with the core of bilin/GAF domain through a salt bridge between two polar residues and aromatic hydrophobic interactions. This β-sheet structure is seen in the Pr state of phytochromes (Figure 10, left). Upon photo-activation by bilin receptors, the β-sheet present in the Pr state changes to α-helix in the Pfr state (Figure 10, right). This, in turn, leads to movement in the helical spine at the dimer interface to stabilize the photo-activated state. Conserved motifs like PR-X-S-F (X is any residue), F/W-X-E/Q, W-A/G-G, present in the tongue region, play a crucial role in stabilizing the interactions.98, 99 Mutating these conserved amino acids hampers the photoconversion.99, 100 The proposed toggle mechanism for light-driven conformational change is observed in bacterial,98, 100-102 cyanobacterial,103 and plant phytochromes.99 The phytochromes in


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these organisms share common homology in the sequence, structure, and function at Pr state and Pfr states (Figure 10).

Figure 10: Phytochrome photo-interconversion between dark-adopted red-light-absorbing Pr state (left) and the light activated far-red light absorbing Pfr state (right). The β-hairpin is present in the Pr state and it interconverts to the α-helix that populates the Pfr state. Figure reproduced with permission from Burgie et al., 2014.99 Copyright 2014 National Academy of Sciences.

OTHER EXAMPLES OF PROTEIN METAMORPHS Conformational transitions have been observed in many other proteins. Although they have not been discussed elaborately in this review, they are briefly mentioned here. In the case of some nuclear receptors such as the retinoic acid receptor, a structural change elicited upon agonist binding is used as a mechanism for controlling gene expression.104 The enzyme cysteine-aspartic protease-6 (caspase-6) shows helix-sheet conversion upon protein substrate binding.105 Cyanobacterial circadian rhythms are controlled by three different proteins KaiA, KaiB and KaiC. Here, KaiB displays a fold switch to activate KaiC and KaiA, so as to initiate the circadian cycle.106


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METHODS TO STUDY CONFORMATIONAL SWITCH IN METAMORPHIC PROTEINS Many proteins and designed peptides show conformational preferences to a particular secondary structure depending on external factors or parameters like concentration of peptide or protein,82 pH,107-109 temperature,110, 111 time,110, 112 ionic strength,69 solvent,113, 114 metal,115, 116 and lipid or micelle concentration.29, 117-124 These external factors can exert pressure on the peptide and allow it to form a stable fold at that condition, by re-establishing local interactions with different partners. By far, structural studies using X-ray crystallography and NMR (nuclear magnetic resonance) have served as the most popular spectroscopic tools to characterize the conformational interconversion seen in metamorphic proteins. In conjunction with protein variants generated through mutagenesis, crystallography and NMR have provided atomistic information on secondary structure transitions and enabled identification of the primary elements responsible for conformational switch. Other spectroscopic methods used to validate structural transitions in metamorphic proteins include electron paramagnetic resonance (EPR),125 circular dichroism (CD),41, 126 Fourier transform infrared (FTIR) spectroscopy,127 and Raman spectroscopy.128, 129 Förster resonance energy transfer (FRET) can also establish the conformational changes; here, fluorescent probes can be strategically introduced such that distances between the fluorophore(s) are different in the two different folds.130 In most metamorphic proteins, the pathway for the conversion from one protein fold to the other protein fold is believed to be direct. If it is not a direct switch, it has to go through intermediates, which have not yet been observed in the vast majority of known metamorphic proteins. If the structural transition follows complete unfolding or an intermediate with lowered


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secondary structure content, these unfolded or intermediate states have not yet been characterized, because the change in fold occurs over very short timescales. Hence, the end state structures are well characterized, and experimental limitations in understanding the intermediates are overcome using MDS. Therefore, we can speculate the pathway of fold transition using MDS. Recent studies on metamorphic proteins like RfaH/RfaH-CTD and human islet amylin polypeptide (hIAPP) using MDS have proved that direct switching between the two folds follows the appearance of intermediates.19, 131, 132 Limited Proteolysis-Selected Reaction Monitoring (LiP-SRM) mass spectrometry can be applied to observe structural changes in biomolecules ranging from small peptides to complex biological samples.133 The LiP-SRM workflow includes proteolysis, which depends on structural features of the protein under native conditions. Samples that are treated to limited proteolysis are separated using gradient chromatography, detected using tandem mass spectrometry, and analyzed through shotgun proteomics. This method has been successfully tested to detect subtle or pronounced conformational changes seen in samples as diverse as amyloid proteins to enzymes involved in carbon metabolism.133


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METAMORPHIC PROTEINS AND PEPTIDE DESIGN USING FIRST PRINCIPLES Peptides play an important role in a variety of biological processes. They serve as attractive tools to obtain a basic understanding of the conformational switch observed in metamorphic proteins.8 Designed peptides are made to preferentially adopt a particular conformation of either α-helix or β-sheet using conformationally constrained amino acids.134-136 However, extrinsic factors can force peptide sequences to form a different fold by forming new intermolecular interactions. This conformational interconversion is prevalently observed in the aggregation phenomenon of amyloid fibrils. Amyloidogenic peptides serve as a good example to understand the importance of hydrophobic and charged residues in the sequence for irreversible conformational conversion.18, 137 Indeed, a 16-residue peptide that was shown to undergo a temperature-driven reversible conformational switch from α-helix to β-sheet110 served as an excellent model system to understand the initial process of amyloid formation. Our focus here, however, will be on soluble peptides that show chameleonic behavior. Overall, while de novo designed sequences with conformationally constrained residues are unlikely to exhibit chameleonic behavior, the possible occurrence of multiple structural states in nature-inspired peptide sequences cannot be ruled out. We discuss a few select examples in this section. One of the earliest chameleonic peptides was a 16-residue amphiphilic sequence from Manfred Mutter and coworkers in 1991, which exhibited a pH-dependent switch from an α-helical oligomer to an antiparallel β-sheet structure.107 Other designed peptide sequences that exhibited a pH, solvent, salt concentration, or temperature dependent conformational switch from β-sheet to α-helical structures include the 17- residue sequences108 from the Kuntz laboratory, and the Aib-


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Gly nucleated octapeptide from the Balaram laboratory.138 The effect of exogenous factors on peptide sequences derived from proteins has also been reported in the case of the 15-residue chameleonic peptide from the CD4-binding domain of HIV gp120113 and peptides from chymotrypsin and liver alcohol dehydrogenase.14 Metal- and redox-mediated conformational switch between α-helix and β-sheet has been reported in designed Zn-binding,109 Cu-binding,116 Zn/Cu-binding115 peptides, and in an oxidation-reduction process involving the Met side chain.126 Chameleonic behavior is uncommon in synthetic membrane-associated peptides derived from natural sequences. Yet, two outstanding and unexpected examples that have been reported so far include the first transmembrane domain of Mycobacteriophage D29 holin peptide (Figure 11A) and the 14-residue peptide of pneumococcus autolysin (Figure 11B). These examples are discussed below. Lytic bacteriophages affect the release of mature progeny phages by the disruption of the bacterial cell wall and membrane. The first step to the physical process of cell lysis involves the formation of holes in the host inner membrane by ‘holin’ proteins produced by the phage.139 Holins can have 1-3 transmembrane domains that adopt a helical structure in the membrane. Holins are quiescently accumulated in the bacterial inner membrane and are maintained in this inactive state by ‘antiholins’ that are co-synthesized in a 2:1 holin: anti-holin ratio.140 Anti-holins are two resides longer than holin proteins. The assembly of mature phage particles triggers the removal of the extra residues from antiholins, converting them to holins.139 Holins form a hole in the host membrane that is sufficiently large to effect progeny phage release.139 In mycobacteriophages, which target various species of mycobacteria, holins are known to occur.139, 141 However, an antiholin has not been identified, which raises the question of how the uncontrolled and premature association of holins is prevented. Our study of the first


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transmembrane domain (TM1) of the Mycobacteriophage D29 holin revealed a surprising conformational switch of TM1 that was lipid dependent.120 At high lipid concentrations, TM1 exists in a helical form, similar to other canonical holins. When more holin proteins are synthesized in the host cytosol and relocated to the inner membrane, the protein: lipid ratio increases in favor of protein. Under these conditions, TM1 undergoes a conformational conversion to a β-hairpin structure, which allows the peptide to aggregate in the membrane and cause membrane destabilization.123 A centrally located Pro-Gly segment that is conserved across mycobacteriophage holins facilitates the reversible conversion of TM1 between the helical and β-hairpin structure, based on the protein: lipid ratio (Figure 11A). A similar lipid-dependent structural conversion between a polyproline conformation (in lipid vesicles) and α-helical conformation (in organic solvents) has been reported for the second transmembrane domain of mycobacteriophage D29 holin.142


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Figure 11: Micelle- triggered conformation change in TM1 (A) and LytA239-252 (B). (A) The centrally located Pro-Gly present in TM1 is important for β-hairpin to α-helix interconversion in low micelle and high micelle concentrations, respectively. This structural interconversion is abolished upon introducing β-hairpin favoring residues (DPro-Gly) or helix favoring residues (Ala-Ala or Aib-Gly) in the central turn region. Panel A is edited and reproduced with permission from Lella et al., 2013120 with permission from The Royal Society of Chemistry. (B) LytA239-252 is present in a β-hairpin form in aqueous conditions (top) and converts to α-helix


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(bottom) when a lipidic (DPC) or detergent (SDS) micelle environment is provided. Panel B is reproduced with permission from Zamora‐Carreras et al., 2015.122 Copyright © 2015 John Wiley and Sons. An interesting case of structural interconversion was reported in an isolated 14-residue sequence of an autolysin from the pathogen Streptococcus pneumoniae. Autolysins possess choline-binding modules (CBMs) that allow these proteins to attach to choline moieties of teichoic and lipoteichoic acids found on the host cell surface. CBMs possess choline-binding repeats (CBRs) that adopt a ββ-solenoid motif. Each strand in this ββ-solenoid motif is constituted by β-hairpins that are about 14 residues in length, and are enriched with aromatic and charged residues.143 Work from the Jiménez group on a CBR of the autolysin LytA (LytA239-252) led to the identification of a structural switch in this 14-residue peptide.122 NMR analysis of LytA239-252 revealed that the peptide adopted a β-hairpin structure in aqueous solutions (Figure 11B). Turn nucleation was achieved at A245-D246 and the non-hydrogen bonding position of the β-hairpin was stabilized by a Lys-Trp and Trp-Tyr interaction.122, 144 Surprisingly, when the peptide was presented with a lipidic environment, it underwent a highly cooperative transition from a β-hairpin to α-helix as the concentration of phosphocholine molecules was increased (Figure 11B). Further characterization of LytA239-252 using NMR and CD showed that the conformational change was facilitated by the interaction of the peptide with phosphocholine micelles and not with the individual phosphocholine molecules. The studies also established that the process of structural conversion in LytA239-252 was reversible. The Jiménez group was also able to demonstrate that the β-hairpin ↔ α-helix conversion could also be elicited in choline and non-choline lipid vesicles, and when non-lipidic micellar system (such as SDS micelles) was provided to the peptide (Figure 11B). As the mechanism of LytA translocation across the


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membrane is not known, the identification of a structural switch allowed the authors to propose that the structural switch in the CBRs of LytA could facilitate this translocation. Overall, the amphipathic and metamorphic nature of the CBR region might assist the LytA molecule in its function of host cell adhesion and pathogenesis.


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CONCLUSIONS Conformational plasticity exhibited by metamorphic proteins appears to be an interesting deviation from Anfinsen’s hypothesis, and poses a challenge to protein structure prediction and de novo design of novel sequences. However, this dual nature of proteins carries an immense applicability in the design of nanoswitches. An increasing number of proteins that exhibit dramatic conformational switch are being identified. These conformational changes can be triggered by various factors including simple external cues such as pH, temperature, and salt concentrations (Figure 12). While metamorphic proteins can be perceived as a conundrum in solving the protein folding problem, we believe that the identification of more naturally occurring protein metamorphs will facilitate the use of these molecules in bionanotechnology as molecular switches.

Figure 12: Summary of intrinsic and extrinsic factors responsible for metamorphic behavior of proteins.


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AUTHOR INFORMATION Corresponding Author *Muralikrishna Lella, E-mail: [email protected]; *Dr. Radhakrishnan Mahalakshmi, E-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. *These authors contributed equally. Funding Sources This work was supported by the Science and Engineering Research Board project SERB/WEA13/2016 to R.M. Notes The authors have no conflict of interest to disclose. ACKNOWLEDGMENT M.L. thanks IISER Bhopal for research fellowship. R.M. is a recipient of the Wellcome Trust/ DBT India Alliance Intermediate Fellowship.


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For Table of Contents Use Only

Metamorphic Proteins: Emergence of dual protein folds from one primary sequence Muralikrishna Lella1,* and Radhakrishnan Mahalakshmi1,*


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Figure 1: Reply to the Paracelsus challenge.


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Figure 2: Evolutionary fold change in the Cro family of transcription factors.


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Figure 3: Conformational flips observed in the transcription factor RfaH.


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Figure 4: Fold change with point mutation.


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Figure 5: Sequential mutagenesis of GA and GB domains.


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Figure 6: Dynamic equilibrium between monomer and dimer with two different folds in Ltn/XCL1.


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Figure 7: Redox-controlled dimerization of CLIC showing the conformational switch in the N-terminal domain.


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Figure 8: Concentration-dependent oligomerization and structural transition of selecase.


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Figure 9: Pore-forming toxins undergo structural transitions upon inserting into the membrane.


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Figure 10: Phytochrome photo-interconversion between dark-adopted red-light-absorbing Pr state (left) and the light activated far-red light absorbing Pfr state (right).


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Figure 11: Micelle- triggered conformation change in TM1 (A) and LytA239-252 (B).


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Figure 12: Summary of intrinsic and extrinsic factors responsible for metamorphic behavior of proteins.


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TOC Graphic 30x26mm (300 x 300 DPI)


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Metamorphic Proteins: Emergence of Dual Protein ... - ACS Publications

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