Aitziber L. Cortajarena

Ligand binding by repeat proteins: natural and designed.

Repeat proteins contain tandem arrays of small structural motifs. As a consequence of this architecture, they adopt non-globular, extended structures that present large, highly specific surfaces for ligand binding. Here we discuss recent advances toward understanding the functional role of this unique modular architecture. We showcase specific examples of natural repeat proteins interacting with diverse ligands and also present examples of designed repeat protein-ligand interactions.

Designed TPR Modules as Novel Anticancer Agents

Molecules specifically designed to modulate protein-protein interactions have tremendous potential as novel therapeutic agents. One important anticancer target is the chaperone Hsp90, whose activity is essential for the folding of many oncogenic proteins, including HER2, IGFIR, AKT, RAF-1, and FLT-3. Here we report the design and characterization of new tetratricopeptide repeat modules, which bind to the C-terminus of Hsp90 with higher affinity and with greater specificity than natural Hsp90-binding co-chaperones. Thus, when these modules are introduced into the cell, they out-compete endogenous co-chaperones for binding, thereby inhibiting Hsp90 function. The effect of Hsp90 inhibition in this fashion is dramatic; HER2 levels are substantially decreased and BT474 HER2 positive breast cancer cells are killed. Our designs thus provide new tools with which to dissect the mechanism of Hsp90-mediated protein folding and also open the door to the development of an entirely new class of anticancer agents.

Ligand binding by TPR domains

Tetratricopeptide repeat (TPR) domains bind specific peptide ligands and are thought to mediate protein–protein interactions in a variety of biological systems. Here we compare peptide ligand‐binding by several different TPR domains. We present specific examples that demonstrate that TPR domains typically undergo little or no structural rearrangement upon ligand binding. Our data suggest that, contrary to a recent proposal, coupled folding and binding is not the common mechanism of ligand recognition by TPR domains.

Singlet Oxygen Generation by the Genetically Encoded Tag miniSOG

The genetically encodable fluorescent tag miniSOG is expected to revolutionize correlative light- and electron microscopy due to its ability to produce singlet oxygen upon light irradiation. The quantum yield of this process was reported as ΦΔ = 0.47 ± 0.05, as derived from miniSOGs ability to photooxidize the fluorescent probe anthracene dipropionic acid (ADPA). In this report, a significantly smaller value of ΦΔ = 0.03 ± 0.01 is obtained by two methods: direct measurement of its phosphorescence at 1275 nm and chemical trapping using uric acid as an alternative probe. We present insight into the photochemistry of miniSOG and ascertain the reasons for the discrepancy in ΦΔ values. We find that miniSOG oxidizes ADPA by both singlet oxygen-dependent and -independent processes. We also find that cumulative irradiation of miniSOG increases its ΦΔ value ~10-fold due to a photoinduced transformation of the protein. This may be the reason why miniSOG outperforms other fluorescent proteins reported to date as singlet oxygen generators.

Structure and stability of designed TPR protein superhelices: unusual crystal packing and implications for natural TPR proteins.

The structure and stability of repeat proteins has been little studied in comparison to the properties of the more familiar globular proteins. Here, the structure and stability of designed tetratricopeptide-repeat (TPR) proteins is described. The TPR is a 34-amino-acid motif which adopts a helix-turn-helix structure and occurs as tandem repeats. The design of a consensus TPR motif (CTPR) has previously been described. Here, the crystal structures and stabilities of proteins that contain eight or 20 identical tandem repeats of the CTPR motif (CTPR8 and CTPR20) are presented. Both CTPR8 and CTPR20 adopt a superhelical overall structure. The structures of the different-length CTPR proteins are compared with each other and with the structures of natural TPR domains. Also, the unusual and perhaps unique crystal-packing interactions resulting in pseudo-infinite crystalline superhelices observed in the different crystal forms of CTPR8 and CTPR20 are discussed. Finally, it is shown that the thermodynamic behavior of CTPR8 and CTPR20 can be predicted from the behavior of other TPRs in this series using an Ising model-based analysis. The designed protein series CTPR2-CTPR20 covers the natural size repertoire of TPR domains and as such is an excellent model system for natural TPR proteins.

Screening Libraries To Identify Proteins with Desired Binding Activities Using a Split-GFP Reassembly Assay

Designer protein modules, which bind specifically to a desired target, have numerous potential applications. One approach to creating such proteins is to construct and screen libraries. Here we present a detailed description of using a split-GFP reassembly assay to screen libraries and identify proteins with novel binding properties. Attractive features of the split-GFP based screen are the absence of false positives and the simplicity, robustness, and ease of automation of the screen. Here, we describe both the construction of a naive protein library, and screening of the library using the split-GFP assay to identify proteins that bind specifically to chosen peptide sequences.

Honeycomb patterned surfaces functionalized with polypeptide sequences for recognition and selective bacterial adhesion

We report on the preparation of functional polymer surfaces with controlled topography by using the breath figures approach. The resulting surfaces prepared from a mixture of a PS-b-PAA diblock copolymer and a homopolymer (PS) exhibit pores that are mainly composed of diblock copolymer whereas the rest of the surface is formed by homopolymer. The formation of a hexagonal assembly of pores was achieved by controlling several parameters during the casting process including relative humidity, composition of the blend and polymer concentration. A selective modification of the pore inner part by using appropriate polypeptide sequences permitted the use of these surfaces as scaffolds for pattern and display of active biomolecules, as ordered templates for specific recognition processes and finally for the micropatterning of bacterial cells.

Crystal structure of a designed tetratricopeptide repeat module in complex with its peptide ligand

Tetratricopeptide repeats (TPRs) are protein domains that mediate key protein–protein interactions in cells. Several TPR domains bind the C‐termini of the chaperones heat shock protein (Hsp)90 and/or Hsp70, and exchange of such binding partners is key for the heat shock response. We have previously described the design of a TPR protein that binds tightly and specifically to the C‐terminus of Hsp90, and in doing so, is able to inhibit chaperone function in vivo. Here we present the X‐ray crystal structure of the designed TPR domain (CTPR390) in complex with its peptide ligand – the C‐terminal residues of Hsp90 (peptide MEEVD). This structure reveals two interesting aspects of the TPR modules. First, a new packing arrangement of 3‐TPR modules is observed. The TPR units stack against each other in an unusual fashion to form infinite superhelices in the crystal. Second, the structure provides insights into the molecular basis of TPR–ligand recognition.

Designed Proteins To Modulate Cellular Networks

A major challenge of protein design is to create useful new proteins that interact specifically with biological targets in living cells. Such binding modules have many potential applications, including the targeted perturbation of protein networks. As a general approach to create such modules, we designed a library with approximately 10(9) different binding specificities based on a small 3-tetratricopeptide repeat (TPR) motif framework. We employed a novel strategy, based on split GFP reassembly, to screen the library for modules with the desired binding specificity. Using this approach, we identified modules that bind tightly and specifically to Dss1, a small human protein that interacts with the tumor suppressor protein BRCA2. We showed that these modules also bind the yeast homologue of Dss1, Sem1. Furthermore, we demonstrated that these modules inhibit Sem1 activity in yeast. This strategy will be generally applicable to make novel genetically encoded tools for systems/synthetic biology applications.

Non-random-coil Behavior as a Consequence of Extensive PPII Structure in the Denatured State

Unfolded proteins may contain a native or nonnative residual structure, which has important implications for the thermodynamics and kinetics of folding, as well as for misfolding and aggregation diseases. However, it has been universally accepted that residual structure should not affect the global size scaling of the denatured chain, which obeys the statistics of random coil polymers. Here we use a single-molecule optical technique--fluorescence correlation spectroscopy--to probe the denatured state of a set of repeat proteins containing an increasing number of identical domains, from 2 to 20. The availability of this set allows us to obtain the scaling law for the unfolded state of these proteins, which turns out to be unusually compact, strongly deviating from random coil statistics. The origin of this unexpected behavior is traced to the presence of an extensive nonnative polyproline II helical structure, which we localize to specific segments of the polypeptide chain. We show that the experimentally observed effects of polyproline II on the size scaling of the denatured state can be well-described by simple polymer models. Our findings suggest a hitherto unforeseen potential of nonnative structure to induce significant compaction of denatured proteins, significantly affecting folding pathways and kinetics.