Comparison of the backbone dynamics of a natural and a consensus designed 3-TPR domain

The tetratricopeptide repeat (TPR) is a 34-amino acid helix-turn-helix motif that occurs in tandem arrays in numerous proteins. Here we compare the backbone dynamics of a natural 3-repeat TPR domain, from the protein UBP, with the behavior of a designed protein CTPR3, which consists of three identical consensus TPR units. Although the three tandem TPR repeats in both CTPR3 and UBP behave as a single unit, with no evidence of independent repeat motions, the data indicate that certain positions in UBP are significantly more flexible than are the corresponding positions in CTPR3. Most of the dynamical changes occur at or adjacent to positions that are involved in intra-repeat packing interactions. These observations lead us to suggest that the three-TPR domain of UBP does not incorporate optimized packing, compared to that seen in the idealized CTPR. The natural TPR domain is not only less stable overall than CTPR3, but also presents increased local flexibility at the positions where the sequences differs from the conserved consensus.     

Calorimetric study of a series of designed repeat proteins: modular structure and modular folding

Repeat proteins comprise tandem arrays of a small structural motif. Their structure is defined and stabilized by interactions between residues that are close in the primary sequence. Several studies have investigated whether their structural modularity translates into modular thermodynamic properties. Tetratricopeptide repeat proteins (TPRs) are a class in which the repeated unit is a 34 amino acid helix-turn-helix motif. In this work, we use differential scanning calorimetry (DSC) to study the equilibrium stability of a series of TPR proteins with different numbers of an identical consensus repeat, from 2 to 20, CTPRa2 to CTPRa20. The DSC data provides direct evidence that the folding/unfolding transition of CTPR proteins does not fit a two-state folding model. Our results confirm and expand earlier studies on TPR proteins, which showed that apparent two-state unfolding curves are better fit by linear statistical mechanics models: 1D Ising models in which each repeat is treated as an independent folding unit.     

Repeat motions and backbone flexibility in designed proteins with different numbers of identical consensus tetratricopeptide repeats

The tetratricopeptide repeat (TPR) is a 34-residue helix-turn-helix motif that occurs as three or more tandem repeats in a wide variety of proteins. We have determined the repeat motions and backbone fluctuations of proteins containing two or three consensus TPR repeats (CTPR2 and CPTR3, respectively) using 15N NMR relaxation measurements. Rotational diffusion tensors calculated from these data for each repeat within each TPR protein indicate that there is a high degree of motional correlation between different repeats in the same protein. This is consistent with the prevailing view that repeat proteins, such as CTPR2 and CTPR3, behave as single cooperatively folded domains. The internal motions of backbone NH groups were determined using the Lipari-Szabo model-free formalism. For most residues, there was a clear separation between the influence of internal motion and the influence of global rotational tumbling on the observed magnetic relaxation. The local internal motions are highly restricted in most of the helical elements, with slightly greater flexibility in the linker elements. Comparisons between CTPR2 and CTPR3 indicate that an addition of a TPR repeat to the C-terminus (before the solvation helix) of CTPR2 slightly reduces the flexibility of the preceding helix.     

TPR重复和ELTR排型:重复的长度变化是一种功能进化的机制

TPR重复最初定义为一个34个氨基酸的蛋白结构重复。本文用PATTINPROT软件对基因库中TPR重复长度多样性进行了分析,发现40和42个氨基酸TPR重复大量存在。含40和42个氨基酸TPR重复序列蛋白的功能分析说明,这些长的TPR重复可以赋予蛋白新的功能。根据以上分析,提出重复序列的长度变化可能是功能进化的一种发生机制。     

Design of stable alpha-helical arrays from an idealized TPR motif

The tetratricopeptide repeat (TPR) is a 34-amino acid alpha-helical motif that occurs in over 300 different proteins. In the different proteins, three to sixteen or more TPR motifs occur in tandem arrays and function to mediate protein-protein interactions. The binding specificity of each TPR protein is different, although the underlying structural motif is the same. Here we describe a statistical approach to the design of an idealized TPR motif. We present the high-resolution X-ray crystal structures (to 1.55 and 1.6 A) of designed TPR proteins and describe their solution properties and stability. A detailed analysis of these structures provides an understanding of the TPR motif, how it is repeated to give helical arrays with different superhelical twists, and how a very stable framework may be constructed for future functional designs.     

Removal of a consensus proline is not sufficient to allow tetratricopeptide repeat oligomerization

Tetratricopeptide repeat (TPR) domains are ubiquitous protein interaction domains that adopt a modular antiparallel array of α‐helices. The TPR fold typically adopts a monomeric state, and consensus TPRs sequences successfully fold into the expected monomeric topology. The versatility of the TPR fold also supports different quaternary structures, which may function as regulatory switches. One example is yeast mitochondrial fission 1 (Fis1) that appears to interconvert between monomer and dimer states in regulating division of peroxisomes and mitochondria. Whether human Fis1 can also interconvert like the yeast molecule is unknown. A TPR consensus proline residue present in human Fis1 is absent in the yeast molecule and, when added, prevents yeast Fis1 dimerization suggesting that the TPR consensus proline might have persisted to prevent TPR oligomerization. Here, we address this question with human Fis1 and the consensus TPR protein CTPR3. We demonstrate that human Fis1 does not form a noncovalent dimer via its TPR domain, despite conditions that favor dimerization of the yeast protein. We also show that the presence of the consensus proline is not sufficient to forbid TPR dimerization. Lastly, an analysis of all available TPR protein structures (22 nonredundant structures, totaling 64 TPRs—42 with the consensus proline and 22 without) revealed that the consensus proline is not necessary for turn formation, but does favor shorter turns. This work suggests the TPR consensus proline is not to prevent oligomerization, but to favor tight turns between repeats.     

Self‐association of TPR domains: Lessons learned from a designed,consensus‐based TPR oligomer

The tetratricopeptide repeat (TPR) motif is a protein–protein interaction module that acts as an organizing centre for complexes regulating a multitude of biological processes. Despite accumulating evidence for the formation of TPR oligomers as an additional level of regulation there is a lack of structural and solution data explaining TPR self‐association. In the present work we characterize the trimeric TPR‐containing protein YbgF, which is linked to the Tol system in Gram‐negative bacteria. By subtracting previously identified TPR consensus residues required for stability of the fold from residues conserved across YbgF homologs, we identified residues involved in oligomerization of the C‐terminal YbgF TPR domain. Crafting these residues, which are located in loop regions between TPR motifs, onto the monomeric consensus TPR protein CTPR3 induced the formation of oligomers. The crystal structure of this engineered oligomer shows an asymmetric trimer where stacking interactions between the introduced tyrosines and displacement of the C‐terminal hydrophilic capping helix, present in most TPR domains, are key to oligomerization. Asymmetric trimerization of the YbgF TPR domain and CTPR3Y3 leads to the formation of higher order oligomers both in the crystal and in solution. However, such open‐ended self‐association does not occur in full‐length YbgF suggesting that the protein's N‐terminal coiled‐coil domain restricts further oligomerization. This interpretation is borne out in experiments where the coiled‐coil domain of YbgF was engineered onto the N‐terminus of CTPR3Y3 and shown to block self‐association beyond trimerization. Our study lays the foundations for understanding the structural basis for TPR domain self‐association and how such self‐association can be regulated in TPR domain‐containing proteins. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

An unexpected extended conformation for the third TPR motif of the peroxin PEX5 from Trypanosoma brucei

A number of helix-rich protein motifs are involved in a variety of critical protein-protein interactions in living cells. One of these is the tetratrico peptide repeat (TPR) motif that is involved, amongst others, in cell cycle regulation, chaperone function and post-translation modifications. So far, these helix-rich TPR motifs have always been observed to be a compact unit of two helices interacting with each other in antiparallel fashion. Here, we describe the structure of the first three TPR-motifs of the peroxin PEX5 from Trypanosoma brucei, the causative agent of sleeping sickness. Peroxins are proteins involved in peroxisome, glycosome and glyoxysome biogenesis. PEX5 is the receptor of the proteins targeted to these organelles by the "peroxisomal targeting signal-1", a C-terminal tripeptide called PTS-1. The first two of the three TPR-motifs of T. brucei PEX5 appear to adopt the canonical antiparallel helix hairpin structure. In contrast, the third TPR motif of PEX5 has a dramatically different conformation in our crystals: the two helices that were supposed to form a hairpin are folded into one single 44 A long continuous helix. Such a conformation has never been observed before for a TPR motif. This raises interesting questions including the potential functional importance of a "jack-knife" conformational change in TPR motifs.     

The highly repetitive region of the Helicobacter pylori CagY protein comprises tandem arrays of an alpha-helical repeat module

The cag-pathogenicity-island-encoded type IV secretion system of Helicobacter pylori functions to translocate the effector protein CagA directly through the plasma membrane of gastric epithelial cells. Similar to other secretion systems, the Cag type IV secretion system elaborates a surface filament structure, which is unusually sheathed by the large cag-pathogenicity-island-encoded protein CagY. CagY is distinguished by unusual amino acid composition and extensive repetitive sequence organised into two defined repeat regions. The second and major repeat region (CagY_rpt2) has a regular disposition of six repetitive motifs, which are subject to deletion and duplication, facilitating the generation of CagY size and phenotypic variants. In this study, we show CagY_rpt2 to comprise two highly thermostable and acid-stable α-helical structural motifs, the most abundant of which (motif A) occurs in tandem arrays of one to six repeats terminally flanked by single copies of the second repeat (motif B). Isolated motifs demonstrate hetero- and homomeric interactions, suggesting a propensity for uniform assembly of discrete structural subunit motifs within the larger CagY_rpt2 structure. Consistent with this, CagY proteins comprising substantially different repeat 2 motif organisations demonstrate equivalent CagA translocation competence, illustrating a remarkable structural and functional tolerance for precise deletion and duplication of motif subunits. We provide the first insight into the structural basis for CagY_rpt2 assembly that accommodates both the variable motif sequence composition and the extensive contraction/expansion of repeat modules within the CagY_rpt2 region.     

Modulation of the multistate folding of designed TPR proteins through intrinsic and extrinsic factors

Tetratricopeptide repeats (TPRs) are a class of all alpha-helical repeat proteins that are comprised of 34-aa helix-turn-helix motifs. These stack together to form nonglobular structures that are stabilized by short-range interactions from residues close in primary sequence. Unlike globular proteins, they have few, if any, long-range nonlocal stabilizing interactions. Several studies on designed TPR proteins have shown that this modular structure is reflected in their folding, that is, modular multistate folding is observed as opposed to two-state folding. Here we show that TPR multistate folding can be suppressed to approximate two-state folding through modulation of intrinsic stability or extrinsic environmental variables. This modulation was investigated by comparing the thermodynamic unfolding under differing buffer regimes of two distinct series of consensus-designed TPR proteins, which possess different intrinsic stabilities. A total of nine proteins of differing sizes and differing consensus TPR motifs were each thermally and chemically denatured and their unfolding monitored using differential scanning calorimetry (DSC) and CD/fluorescence, respectively. Analyses of both the DSC and chemical denaturation data show that reducing the total stability of each protein and repeat units leads to observable two-state unfolding. These data highlight the intimate link between global and intrinsic repeat stability that governs whether folding proceeds by an observably two-state mechanism, or whether partial unfolding yields stable intermediate structures which retain sufficient stability to be populated at equilibrium.     

The Assembly and Intermolecular Properties of the Hsp70-Tomm34-Hsp90 Molecular Chaperone Complex

Maintenance of protein homeostasis by molecular chaperones Hsp70 and Hsp90 requires their spatial and functional coordination. The cooperation of Hsp70 and Hsp90 is influenced by their interaction with the network of co-chaperone proteins, some of which contain tetratricopeptide repeat (TPR) domains. Critical to these interactions are TPR domains that target co-chaperone binding to the EEVD-COOH motif that terminates Hsp70/Hsp90. Recently, the two-TPR domain-containing protein, Tomm34, was reported to bind both Hsp70 and Hsp90. Here we characterize the structural basis of Tomm34-Hsp70/Hsp90 interactions. Using multiple methods, including pull-down assays, fluorescence polarization, hydrogen/deuterium exchange, and site-directed mutagenesis, we defined the binding activities and specificities of Tomm34 TPR domains toward Hsp70 and Hsp90. We found that Tomm34 TPR1 domain specifically binds Hsp70. This interaction is partly mediated by a non-canonical TPR1 two-carboxylate clamp and is strengthened by so far unidentified additional intermolecular contacts. The two-carboxylate clamp of the isolated TPR2 domain has affinity for both chaperones, but as part of the full-length Tomm34 protein, the TPR2 domain binds specifically Hsp90. These binding properties of Tomm34 TPR domains thus enable simultaneous binding of Hsp70 and Hsp90. Importantly, we provide evidence for the existence of an Hsp70-Tomm34-Hsp90 tripartite complex. In addition, we defined the basic conformational demands of the Tomm34-Hsp90 interaction. These results suggest that Tomm34 represents a novel scaffolding co-chaperone of Hsp70 and Hsp90, which may facilitate Hsp70/Hsp90 cooperation during protein folding.     

The crystal structure of NlpI. A prokaryotic tetratricopeptide repeat protein with a globular fold

There are several different families of repeat proteins. In each, a distinct structural motif is repeated in tandem to generate an elongated structure. The nonglobular, extended structures that result are particularly well suited to present a large surface area and to function as interaction domains. Many repeat proteins have been demonstrated experimentally to fold and function as independent domains. In tetratricopeptide (TPR) repeats, the repeat unit is a helix-turn-helix motif. The majority of TPR motifs occur as three to over 12 tandem repeats in different proteins. The majority of TPR structures in the Protein Data Bank are of isolated domains. Here we present the high-resolution structure of NlpI, the first structure of a complete TPR-containing protein. We show that in this instance the TPR motifs do not fold and function as an independent domain, but are fully integrated into the three-dimensional structure of a globular protein. The NlpI structure is also the first TPR structure from a prokaryote. It is of particular interest because it is a membrane-associated protein, and mutations in it alter septation and virulence.     

Thermodynamics, kinetics, and salt dependence of folding of YopM, a large leucine-rich repeat protein

Small globular proteins have many contacts between residues that are distant in primary sequence. These contacts create a complex network between sequence-distant segments of secondary structure, which may be expected to promote the cooperative folding of globular proteins. Although repeat proteins, which are composed of tandem modular units, lack sequence-distant contacts, several of considerable length have been shown to undergo cooperative two-state folding. To explore the limits of cooperativity in repeat proteins, we have studied the unfolding of YopM, a leucine-rich repeat (LRR) protein of over 400 residues. Despite its large size and modular architecture (15 repeats), YopM equilibrium unfolding is highly cooperative, and shows a very strong dependence on the concentration of urea. In contrast, kinetic studies of YopM folding indicate a mechanism that includes one or more transient intermediates. The urea dependence of the folding and unfolding rates suggests a relatively small transition state ensemble. As with the urea dependence, we have found an extreme dependence of the free energy of unfolding on the concentration of salt. This salt dependence likely results from general screening of a large number of unfavorable columbic interactions in the folded state, rather than from specific cation binding.     

Self-assembly of repeat proteins: Concepts and design of new interfaces

In nature, assembled protein structures offer the most complex functional structures. The understanding of the mechanisms ruling protein–protein interactions opens the door to manipulate protein assemblies in a rational way. Proteins are versatile scaffolds with great potential as tools in nanotechnology and biomedicine because of their chemical, structural, and functional versatility. Currently, bottom-up self-assembly based on biomolecular interactions of small and well-defined components, is an attractive approach to biomolecular engineering and biomaterial design. Specifically, repeat proteins are simplified systems for this purpose.In this work, we provide an overview of fundamental concepts of the design of new protein interfaces. We describe an experimental approach to form higher order architectures by a bottom-up assembly of repeated building blocks. For this purpose, we use designed consensus tetratricopeptide repeat proteins (CTPRs). CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelices. Introducing a novel interface along the CTPR superhelix allows two CTPR molecules to assemble into protein nanotubes. We apply three approaches to form protein nanotubes: electrostatic interactions, hydrophobic interactions, and π-π interactions. We isolate and characterize the stability and shape of the formed dimers and analyze the nanotube formation considering the energy of the interaction and the structure in the three different models. These studies provide insights into the design of novel protein interfaces for the control of the assembly into more complex structures, which will open the door to the rational design of nanostructures and ordered materials for many potential applications in nanotechnology.     

Modulating repeat protein stability: the effect of individual helix stability on the collective behavior of the ensemble

Repeat proteins are tandem arrays of a small structural motif, in which tertiary structure is stabilized by interactions within a repeat and between neighboring repeats. Several studies have shown that this modular structure is manifest in modular thermodynamic properties. Specifically, the global stability of a repeat protein can be described by simple linear models, considering only two parameters: the stability of the individual repeated units (H) and the coupling interaction between the units (J). If the repeat units are identical, single values of H and J, together with the number of repeated units, is sufficient to completely describe the thermodynamic behavior of any protein within a series. In this work, we demonstrate how the global stability of a repeat protein can be changed, in a predictable fashion, by modifying only the H parameter. Taking a previously characterized series of consensus tetratricopeptide repeats (TPR) (CTPRa) proteins, we introduced mutations into the basic repeating unit, such that the stability of the individual repeat unit was increased, but its interaction with neighboring units was unchanged. In other words, we increased H but kept J constant. We demonstrated that the denaturation curves for a series of such repeat proteins can be fit and additional curves can be predicted by the one-dimensional Ising model in which only H has changed from the original fit for the CTPRa series. Our results show that we can significantly increase the stability of a repeat protein by rationally increasing the stability of the units (H), whereas the interaction between repeats (J) remains unchanged.