Eva S. Cobos

PDZ domains are binding modules mostly involved in cell signaling and cell–cell junctions. These domains are able to recognize a wide variety of natural targets and, among the PDZ partners, viruses have been discovered to interact with their host via a PDZ domain. With such an array of relevant and diverse interactions, PDZ binding specificity has been thoroughly studied and a traditional classification has grouped PDZ domains in three major specificity classes. In this work, we have selected four human PDZ domains covering the three canonical specificity-class binding mode and a set of their corresponding binders, including host/natural, viral and designed PDZ motifs. Through calorimetric techniques, we have covered the entire cross interactions between the selected PDZ domains and partners. The results indicate a rather basic specificity in each PDZ domain, with two of the domains that bind their cognate and some non-cognate ligands and the two other domains that basically bind their cognate partners. On the other hand, the host partners mostly bind their corresponding PDZ domain and, interestingly, the viral ligands are able to bind most of the studied PDZ domains, even those not previously described. Some viruses may have evolved to use of the ability of the PDZ fold to bind multiple targets, with resulting affinities for the virus–host interactions that are, in some cases, higher than for host–host interactions.

In 1972 Christian B. Anfinsen received the Nobel Prize in Chemistry for “…his work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation.” The understanding of this principle is crucial for physical biochemistry students, since protein folding studies, bio‐computing sciences and protein design approaches are founded on such a well‐demonstrated connection. Herein, we describe a detailed and easy‐to‐follow experiment to reproduce the most relevant assays carried out at Anfinsen's laboratory in the 60s. This experiment provides students with a platform to interpret by themselves the structural and kinetic experiments conceived to understand the protein folding problem. In addition, this three‐day experiment brings students a nice opportunity for protein manipulation as well as for the setting up of spectroscopic and chromatographic techniques. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):262–269, 2018.

The term biocalorimetry refers to the application of calorimetry to the study of the energetics of biological processes. Current high-sensitivity commercially available calorimeters offer the possibility of using small sample volumes of very dilute solutions and are able to directly measure very small amounts of heat, which allow the analysis of the thermodynamics of non-covalent interactions, such as those involved in biological macromolecules. This chapter presents an overview of the most relevant applications of DSC to protein solution studies. A survey of the main theoretical models to interpret and analyze protein calorimetric thermograms is described, together with an evaluation of their applicability. Here the main goal of DSC is to characterize protein stability and analyze protein thermal unfolding. From the DSC measurements all the meaningful thermodynamic parameters of such processes, together with the temperature-dependent population of the initial, final and possible intermediate states can be determined. The wider possibilities of DSC to investigate the effect of protein self-association as well as that of the presence of protein ligands during the thermal processes are also described. Finally, when the unfolding process does not occur under equilibrium, DSC is also a very useful technique to study protein denaturation through a kinetic approach.

YAP is a WW domain-containing effector of the Hippo tumor suppressor pathway, and the object of heightened interest as a potent oncogene and stemness factor. YAP has two major isoforms that differ in the number of WW domains they harbor. Elucidating the degree of co-operation between these WW domains is important for a full understanding of the molecular function of YAP. We present here a detailed biophysical study of the structural stability and binding properties of the two YAP WW domains aimed at investigating the relationship between both domains in terms of structural stability and partner recognition. We have carried out a calorimetric study of the structural stability of the two YAP WW domains, both isolated and in a tandem configuration, and their interaction with a set of functionally relevant ligands derived from PTCH1 and LATS kinases. We find that the two YAP WW domains behave as independent units with different binding preferences, suggesting that the presence of the second WW domain might contribute to modulate target recognition between the two YAP isoforms. Analysis of structural models and phage-display studies indicate that electrostatic interactions play a critical role in binding specificity. Together, these results are relevant to understand of YAP function and open the door to the design of highly specific ligands of interest to delineate the functional role of each WW domain in YAP signaling.

During HIV-1 fusion to the host cell membrane, the N-terminal heptad repeat (NHR) and the C-terminal heptad repeat (CHR) of the envelope subunit gp41 become transiently exposed and accessible to fusion inhibitors or Abs. In this process, the NHR region adopts a trimeric coiled-coil conformation that can be a target for therapeutic intervention. Here, we present an approach to rationally design single-chain protein constructs that mimic the NHR coiled-coil surface. The proteins were built by connecting with short loops two parallel NHR helices and an antiparallel one with the inverse sequence followed by engineering of stabilizing interactions. The constructs were expressed in Escherichia coli, purified with high yield, and folded as highly stable helical coiled coils. The crystal structure of one of the constructs confirmed the predicted fold and its ability to accurately mimic an exposed gp41 NHR surface. These single-chain proteins bound to synthetic CHR peptides with very high affinity, and furthermore, they showed broad inhibitory activity of HIV-1 fusion on various pseudoviruses and primary isolates. Significance The envelope subunit gp41 is an attractive target for therapeutic intervention against HIV-1. Interfering with the interaction between the heptad-repeat regions of gp41 is a promising approach to inhibit HIV-1 fusion to the host cell membrane. Here, we present an alternative rational design and protein-engineering approach to produce highly stable single-chain proteins that accurately mimic the trimeric coiled-coil surface of the gp41 N-terminal heptad repeat. This approach has a strong potential for development to HIV-1 drugs, vaccines, or microbicides and could be extendable to the design of proteins interfering with other types of coiled-coil interactions.

The modulation of binding affinities and specificities by post-translational modifications located out from the binding pocket of the third PDZ domain of PSD-95 (PDZ3) has been reported recently. It is achieved through an intra-domain electrostatic network involving some charged residues in the β2–β3 loop (were a succinimide modification occurs), the α3 helix (an extra-structural element that links the PDZ3 domain with the following SH3 domain in PSD-95, and contains the phosphorylation target Tyr397), and the ligand peptide. Here, we have investigated the main structural and thermodynamic aspects that these structural elements and their related post-translational modifications display in the folding/misfolding pathway of PDZ3 by means of site-directed mutagenesis combined with calorimetry and spectroscopy. We have found that, although all the assayed mutations generate proteins more prone to aggregation than the wild-type PDZ3, those directly affecting the α3 helix, like the E401R substitution or the truncation of the whole α3 helix, increase the population of the DSC-detected intermediate state and the misfolding kinetics, by organizing the supramacromolecular structures at the expense of the two β-sheets present in the PDZ3 fold. However, those mutations affecting the β2–β3 loop, included into the prone-to-aggregation region composed by a single β-sheet comprising β2 to β4 chains, stabilize the trimeric intermediate previously shown in the wild-type PDZ3 and slow-down aggregation, also making it partly reversible. These results strongly suggest that the α3 helix protects to some extent the PDZ3 domain core from misfolding. This might well constitute the first example where an extra-element, intended to link the PDZ3 domain to the following SH3 in PSD-95 and in other members of the MAGUK family, not only regulates the binding abilities of this domain but it also protects PDZ3 from misfolding and aggregation. The influence of the post-translational modifications in this regulatory mechanism is also discussed.

GYF domains are small polyproline-recognition modules adopting a structural arrangement consisting of a single α-helix packed against a small β-sheet. Although most families of proline-rich recognition modules have been extensively characterized in terms of function, structure, or conformational flexibility, little is known about GYF domain functionality and folding. We have undertaken the thermodynamic characterization of the unfolding of CD2BP2-GYF domain by combining differential scanning calorimetry and circular dichroism under different pH conditions. The experimental data can be well-described in terms of a two-state equilibrium, although an unusually high heat capacity of the native state reflects a considerable conformational flexibility and dynamics of CD2BP2-GYF domain. In addition, the normalized thermodynamic parameters of unfolding (enthalpy, entropy and heat capacity) are roughly a factor of two greater than expected. In contrast, stability curves reveal an ordinary unfolding behavior of CD2BP2-GYF domain in terms of Gibbs energies, incurring thus unusually strong enthalpy–entropy compensation. This phenomenon, previously described as “thermodynamic homeostasis”, has been associated in different examples to the contribution of occluded water (solvent) molecules into the protein structure. By means of CASTp server, we have found seven cavities/pockets scattered throughout of the CD2BP2-GYF structure, each able to harbor at least one water molecule. This structural feature provides rationalization for the atypical enthalpy values observed for CD2BP2-GYF because each water molecule is able to organize an extra amount of hydrogen bonds in the native state. In addition, these bound waters increase the vibrational entropy of the protein, which could also be responsible for an increase in protein flexibility and may thus fully explain the homeostatic behavior experimentally observed.

8‐Anilino‐1‐naphthalenesulfonic acid (ANS) is a popular fluorescence probe, broadly used for the analysis of proteins, but the nature of its interaction with proteins and the high increase in the fluorescence intensity that takes place upon such process are still unclear. In the last few years, isothermal titration calorimetry has been used to characterize the nature of the interaction of this dye with proteins. The analysis of the binding isotherms of these studies has not considered the dimerization equilibrium of ANS, which is pH dependent, and it can result in serious errors in the data analysis. In the present work we have developed a suitable data analysis by which this process is taken into account. To study the binding of the dye to proteins at different pH values, we have used the Abl‐SH3 domain. Our results suggest that at pH 3 and 5, where the dimerization of the ANS is important, electrostatic interactions are significant for the binding of ANS to the Abl‐SH3 domain. However, at pH 7, ANS behaves mostly as monomer and the interaction with the protein is mainly hydrophobic. The pH dependent behavior of the ANS binding to proteins can be explained in terms of ionization states of both, the protein and the ANS. Copyright © 2010 John Wiley & Sons, Ltd.