Kaushik Dutta

Mapping structural interactions using in-cell NMR spectroscopy (STINT-NMR)

We describe a high-throughput in-cell nuclear magnetic resonance (NMR)-based method for mapping the structural changes that accompany protein-protein interactions (STINT-NMR). The method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring the protein interactions using in-cell NMR spectroscopy. The resulting spectra provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution.

In-cell NMR for protein-protein interactions (STINT-NMR).

We describe an in-cell NMR-based method for mapping the structural interactions (STINT-NMR) that underlie protein-protein complex formation. This method entails sequentially expressing two (or more) proteins within a single bacterial cell in a time-controlled manner and monitoring their interactions using in-cell NMR spectroscopy. The resulting NMR data provide a complete titration of the interaction and define structural details of the interacting surfaces at atomic resolution. Unlike the case where interacting proteins are simultaneously overexpressed in the labeled medium, in STINT-NMR the spectral complexity is minimized because only the target protein is labeled with NMR-active nuclei, which leaves the interactor protein(s) cryptic. This method can be combined with genetic and molecular screens to provide a structural foundation for proteomic studies. The protocol takes 4 d from the initial transformation of the bacterial cells to the acquisition of the NMR spectra.

pH-induced folding of an apoptotic coiled coil

Par‐4 is a 38‐kD protein pivotal to the apoptotic pathways of various cell types, most notably prostate cells and neurons, where it has been linked to prostate cancer and various neurodegenerative disorders including Alzheimers and Huntingtons diseases and HIV encephalitis. The C‐terminal region of Par‐4 is responsible for homodimerization and the ability of Par‐4 to interact with proposed effector molecules. In this study, we show that the C‐terminal 47 residues of Par‐4 are natively unfolded at physiological pH and temperature. Evidence is rapidly accumulating that natively unfolded proteins play an important role in various cellular functions and signaling pathways, and that folding can often be induced on complexation with effector molecules or alteration of environment. Here we use primarily CD studies to show that changes in the environment, particularly pH and temperature, can induce the Par‐4 C terminus to form a self‐associated coiled coil.

The molecular mechanism of nuclear transport revealed by atomic-scale measurements

Nuclear pore complexes (NPCs) form a selective filter that allows the rapid passage of transport factors (TFs) and their cargoes across the nuclear envelope, while blocking the passage of other macromolecules. Intrinsically disordered proteins (IDPs) containing phenylalanyl-glycyl (FG)-rich repeats line the pore and interact with TFs. However, the reason that transport can be both fast and specific remains undetermined, through lack of atomic-scale information on the behavior of FGs and their interaction with TFs. We used nuclear magnetic resonance spectroscopy to address these issues. We show that FG repeats are highly dynamic IDPs, stabilized by the cellular environment. Fast transport of TFs is supported because the rapid motion of FG motifs allows them to exchange on and off TFs extremely quickly through transient interactions. Because TFs uniquely carry multiple pockets for FG repeats, only they can form the many frequent interactions needed for specific passage between FG repeats to cross the NPC. DOI: http://dx.doi.org/10.7554/eLife.10027.001

A Full-Length Group 1 Bacterial Sigma Factor Adopts a Compact Structure Incompatible with DNA Binding

The sigma factors are the key regulators of bacterial transcription initiation. Through direct read-out of promoter DNA sequence, they recruit the core RNA polymerase to sites of initiation, thereby dictating the RNA polymerase promoter-specificity. The group 1 sigma factors, which direct the vast majority of transcription initiation during log phase growth and are essential for viability, are autoregulated by an N-terminal sequence known as sigma1.1. We report the solution structure of Thermotoga maritima sigmaA sigma1.1. We additionally demonstrate by using chemical crosslinking strategies that sigma1.1 is in close proximity to the promoter recognition domains of sigmaA. We therefore propose that sigma1.1 autoinhibits promoter DNA binding of free sigmaA by stabilizing a compact organization of the sigma factor domains that is unable to bind DNA.

The regions of securin and cyclin B proteins recognized by the ubiquitination machinery are natively unfolded

The proteins securin and cyclin B are destroyed in mitosis by the ubiquitin/proteasome system. This destruction is important to mitotic progression. The N‐terminal regions of these proteins contain the sequence features recognized by the ubiquitination system. We have demonstrated using circular dichroism and 1‐D and 2‐D nuclear magnetic resonance that these rather substantial regions are natively unfolded. Based on these findings, we propose a model that helps to explain previously enigmatic observations.

The Alternatively Spliced Acid Box Region Plays a Key Role in FGF Receptor Autoinhibition

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human malignancies necessitating multiple layers of self-regulatory control mechanisms. Fibroblast growth factor receptor (FGFR) autoinhibition mediated by the alternatively spliced immunoglobulin (Ig) domain 1 (D1) and the acid box (AB)-containing linker between D1 and Ig domain 2 (D2) serves as the first line of defense to minimize inadvertent FGF signaling. In this report, nuclear magnetic resonance and surface plasmon resonance spectroscopy are used to demonstrate that the AB subregion of FGFR electrostatically engages the heparan sulfate (HS)-binding site on the D2 domain in cis to directly suppress HS-binding affinity of FGFR. Furthermore, the cis electrostatic interaction sterically autoinhibits ligand-binding affinity of FGFR because of the close proximity of HS-binding and primary ligand-binding sites on the D2 domain. These data, together with the strong amino acid sequence conservation of the AB subregion among FGFR orthologs, highlight the universal role of the AB subregion in FGFR autoinhibition.

Local control of a disorder-order transition in 4E-BP1 underpins regulation of translation via eIF4E.

The molecular mechanism underpinning regulation of eukaryotic translation initiation factor eIF4E by 4E-BP1 has remained unclear. We use isothermal calorimetry, circular dichroism, NMR, and computational modeling to analyze how the structure of the eIF4E-binding domain of 4E-BP1 determines its affinity for the dorsal face of eIF4E and thus the ability of this regulator to act as a competitive inhibitor. This work identifies the key role of solvent-facing amino acids in 4E-BP1 that are not directly engaged in interactions with eIF4E. These amino acid residues influence the propensity of the natively unfolded binding motif to fold into a conformation, including a stretch of α-helix, that is required for tight binding to eIF4E. In so doing, they contribute to a free energy landscape for 4E-BP1 folding that is poised so that phosphorylation of S65 at the C-terminal end of the helical region can modulate the propensity of folding, and thus regulate the overall free energy of 4E-BP1 binding to eIF4E, over a physiologically significant range. Thus, phosphorylation acts as an intramolecular structural modulator that biases the free energy landscape for the disorder–order transition of 4E-BP1 by destabilizing the α-helix to favor the unfolded form that cannot bind eIF4E. This type of order–disorder regulatory mechanism is likely to be relevant to other intermolecular regulatory phenomena in the cell.

Slide-and-exchange mechanism for rapid and selective transport through the nuclear pore complex

Significance The nuclear pore complex (NPC) mediates the trafficking of macromolecules in and out of the nucleus of eukaryotic cells. Here, we characterize how transport factors diffuse rapidly through multiple layers of disordered phenylalanine-glycine (FG) repeat domains lining the NPC. Transport factors interact with FG repeats through a dynamic sliding motion, enabling faster translocation through the NPC than that attainable by a two-state binding mechanism as well as effectively blocking the passage of large macromolecules that do not bind to transport factors. Thus, the NPC exemplifies a dynamic system in living cells, the function of which depends on protein–protein interactions that are transient on the one hand, and highly specific on the other. Nucleocytoplasmic transport is mediated by the interaction of transport factors (TFs) with disordered phenylalanine-glycine (FG) repeats that fill the central channel of the nuclear pore complex (NPC). However, the mechanism by which TFs rapidly diffuse through multiple FG repeats without compromising NPC selectivity is not yet fully understood. In this study, we build on our recent NMR investigations showing that FG repeats are highly dynamic, flexible, and rapidly exchanging among TF interaction sites. We use unbiased long timescale all-atom simulations on the Anton supercomputer, combined with extensive enhanced sampling simulations and NMR experiments, to characterize the thermodynamic and kinetic properties of FG repeats and their interaction with a model transport factor. Both the simulations and experimental data indicate that FG repeats are highly dynamic random coils, lack intrachain interactions, and exhibit significant entropically driven resistance to spatial confinement. We show that the FG motifs reversibly slide in and out of multiple TF interaction sites, transitioning rapidly between a strongly interacting state and a weakly interacting state, rather than undergoing a much slower transition between strongly interacting and completely noninteracting (unbound) states. In the weakly interacting state, FG motifs can be more easily displaced by other competing FG motifs, providing a simple mechanism for rapid exchange of TF/FG motif contacts during transport. This slide-and-exchange mechanism highlights the direct role of the disorder within FG repeats in nucleocytoplasmic transport, and resolves the apparent conflict between the selectivity and speed of transport.

N-terminal cysteinyl proteins can be prepared using thrombin cleavage.

Expressed protein ligation – which allows native proteins to be selectively linked together by a normal peptide bond in an aqueous environment – has emerged as a powerful technique. The technique requires the formation of a C‐terminal α‐thioester and an N‐terminal Cys. An N‐terminal Cys can be formed by enzymatic cleavage, commonly using the Factor Xa and TEV proteases. We show that thrombin can be used for the formation of N‐terminal Cys, providing another choice of reagents for expressed protein ligation. Proteins with N‐terminal Cys can be obtained by the convenient modification of vectors with the putative thrombin cleavage site LVPRG to LVPRC. Two example protein domains (Csk and Abl tyrosine kinase domain) with N‐terminal Cys are demonstrated using this method. The use of thrombin protease to generate N‐terminal Cys overcomes some of the limitations of existing methods, making it generally useful for expressed protein ligation and other biotechnological applications.