Felipe Merino

OCT4: dynamic DNA binding pioneers stem cell pluripotency.

OCT4 was discovered more than two decades ago as a transcription factor specific to early embryonic development. Early studies with OCT4 were descriptive and looked at determining the functional roles of OCT4 in the embryo as well as in pluripotent cell lines derived from embryos. Later studies showed that OCT4 was one of the transcription factors in the four-factor cocktail required for reprogramming somatic cells into induced pluripotent stem cells (iPSCs) and that it is the only factor that cannot be substituted in this process by other members of the same protein family. In recent years, OCT4 has emerged as a master regulator of the induction and maintenance of cellular pluripotency, with crucial roles in the early stages of differentiation. Currently, mechanistic studies look at elucidating the molecular details of how OCT4 contributes to establishing selective gene expression programs that define different developmental stages of pluripotent cells. OCT4 belongs to the POU family of proteins, which have two conserved DNA-binding domains connected by a variable linker region. The functions of OCT4 depend on its ability to recognize and bind to DNA regulatory regions alone or in cooperation with other transcription factors and on its capacity to recruit other factors required to regulate the expression of specific sets of genes. Undoubtedly, future iPSC-based applications in regenerative medicine will benefit from understanding how OCT4 functions. Here we provide an integrated view of OCT4 research conducted to date by reviewing the different functional roles for OCT4 and discussing the current progress in understanding their underlying molecular mechanisms. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.

The ADP-dependent sugar kinase family: kinetic and evolutionary aspects.

Some archaea of the Euryarchaeota present a unique version of the Embden–Meyerhof pathway where glucose and fructose‐6‐phosphate are phoshporylated using ADP instead of ATP as the phosphoryl donor. These are the only ADP‐dependent kinases known to date. Although initially they were believed to represent a new protein family, they can be classified as members of the ribokinase superfamily, which also include several ATP‐dependent kinases. As they were first identified in members of the thermococcales it was proposed that the presence of these ADP‐dependent kinases is an adaptation to high temperatures. Later, homologs of these enzymes were identified in the genomes of mesophilic and thermophilic methanogenic archaea and even in the genomes of higher eukaryotes, suggesting that the presence of these proteins is not related to the hyperthermophilic life. The ADP‐dependent kinases are very restrictive to their ligands being unable to use triphosphorylated nucleotides such as ATP. However, it has been shown that they can bind ATP by competition kinetic experiments. The hyperthermophilic methanogenic archaeon Methanocaldococcus jannaschii has a homolog of these genes, which can phosphorylate glucose and fructose‐6‐phosphate. For this reason, it was proposed as an ancestral form for the family. However, recent studies have shown that the ancestral activity in the group is glucokinase, and a combination of gene duplication and lateral gene transfer could have originated the two paralogs in this member of the Euryarchaeota. Interestingly, based on structural comparisons made within the superfamily it has been suggested that the ADP‐dependent kinases are the newest in the group. In several members of the superfamily, the presence of divalent metal cations has been shown to be crucial for catalysis, so its role in the ADP‐dependent family was investigated through molecular dynamics. The simulation shows that, in fact, the metal coordinates the catalytic ensemble and interacts with crucial residues for catalysis.

Membrane insertion of a Tc toxin in near-atomic detail

Tc toxins from pathogenic bacteria use a special syringe-like mechanism to perforate the host cell membrane and inject a deadly enzyme into the host cytosol. The molecular mechanism of this unusual injection system is poorly understood. Using electron cryomicroscopy, we determined the structure of TcdA1 from Photorhabdus luminescens embedded in lipid nanodiscs. In our structure, compared with the previous structure of TcdA1 in the prepore state, the transmembrane helices rearrange in the membrane and open the initially closed pore. However, the helices do not span the complete membrane; instead, the loops connecting the helices form the rim of the funnel. Lipid head groups reach into the space between the loops and consequently stabilize the pore conformation. The linker domain is folded and packed into a pocket formed by the other domains of the toxin, thereby considerably contributing to stabilization of the pore state.

High-resolution single particle analysis from electron cryo-microscopy images using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 Å structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

ADP-dependent 6-phosphofructokinase from Pyrococcus horikoshii OT3: structure determination and biochemical characterization of PH1645.

Some hyperthermophilic archaea use a modified glycolytic pathway that employs an ADP-dependent glucokinase (ADP-GK) and an ADP-dependent phosphofructokinase (ADP-PFK) or, in the case of Methanococcus jannaschii, a bifunctional ADP-dependent glucophosphofructokinase (ADP-GK/PFK). The crystal structures of three ADP-GKs have been determined. However, there is no structural information available for ADP-PFKs or the ADP-GK/PFK. Here, we present the first crystal structure of an ADP-PFK from Pyrococcus horikoshii OT3 (PhPFK) in both apo- and AMP-bound forms determined to 2.0-Å and 1.9-Å resolution, respectively, along with biochemical characterization of the enzyme. The overall structure of PhPFK maintains a similar large and small α/β domain structure seen in the ADP-GK structures. A large conformational change accompanies binding of phosphoryl donor, acceptor, or both, in all members of the ribokinase superfamily characterized thus far, which is believed to be critical to enzyme function. Surprisingly, no such conformational change was observed in the AMP-bound PhPFK structure compared with the apo structure. Through comprehensive site-directed mutagenesis of the substrate binding pocket we identified residues that were critical for both substrate recognition and the phosphotransfer reaction. The catalytic residues and many of the substrate binding residues are conserved between PhPFK and ADP-GKs; however, four key residues differ in the sugar-binding pocket, which we have shown determine the sugar-binding specificity. Using these results we were able to engineer a mutant PhPFK that mimics the ADP-GK/PFK and is able to phosphorylate both fructose 6-phosphate and glucose.

Cooperative DNA Recognition Modulated by an Interplay between Protein-Protein Interactions and DNA-Mediated Allostery

Highly specific transcriptional regulation depends on the cooperative association of transcription factors into enhanceosomes. Usually, their DNA-binding cooperativity originates from either direct interactions or DNA-mediated allostery. Here, we performed unbiased molecular simulations followed by simulations of protein-DNA unbinding and free energy profiling to study the cooperative DNA recognition by OCT4 and SOX2, key components of enhanceosomes in pluripotent cells. We found that SOX2 influences the orientation and dynamics of the DNA-bound configuration of OCT4. In addition SOX2 modifies the unbinding free energy profiles of both DNA-binding domains of OCT4, the POU specific and POU homeodomain, despite interacting directly only with the first. Thus, we demonstrate that the OCT4-SOX2 cooperativity is modulated by an interplay between protein-protein interactions and DNA-mediated allostery. Further, we estimated the change in OCT4-DNA binding free energy due to the cooperativity with SOX2, observed a good agreement with experimental measurements, and found that SOX2 affects the relative DNA-binding strength of the two OCT4 domains. Based on these findings, we propose that available interaction partners in different biological contexts modulate the DNA exploration routes of multi-domain transcription factors such as OCT4. We consider the OCT4-SOX2 cooperativity as a paradigm of how specificity of transcriptional regulation is achieved through concerted modulation of protein-DNA recognition by different types of interactions.

Electron Cryo-microscopy as a Tool for Structure-Based Drug Development

For decades, X-ray crystallography and NMR have been the most important techniques for studying the atomic structure of macromolecules. However, as a result of size, instability, low yield, and other factors, many macromolecules are difficult to crystallize or unsuitable for NMR studies. Electron cryo-microscopy (cryo-EM) does not depend on crystals and has therefore been the method of choice for many macromolecular complexes that cannot be crystallized, but atomic resolution has mostly been beyond its reach. A new generation of detectors that are capable of sensing directly the incident electrons has recently revolutionized the field, with structures of macromolecules now routinely being solved to near-atomic resolution. In this review, we summarize some of the most recent examples of high-resolution cryo-EM structures. We put particular emphasis on proteins with pharmacological relevance that have traditionally been inaccessible to crystallography. Furthermore, we discuss examples where interactions with small molecules have been fully characterized at atomic resolution. Finally, we stress the current limits of cryo-EM, and methodological issues related to its usage as a tool for drug development.

Specificity evolution of the ADP‐dependent sugar kinase family –in silico studies of the glucokinase/phosphofructokinase bifunctional enzyme from Methanocaldococcus jannaschii

In several archaea of the Euryarchaeota, the glycolytic flux proceeds through a modified version of the Embden–Meyerhof pathway, where the phosphofructokinase and glucokinase enzymes use ADP as the phosphoryl donor. These enzymes are homologous to each other. In the hyperthermophilic methanogenic archaeon Methanocaldococcus jannaschii, it has been possible to identify only one homolog for these enzymes, which shows both ADP‐dependent glucokinase and phosphofructokinase activity. This enzyme has been proposed as an ancestral form in this family. In this work we studied the evolution of this protein family using the Bayesian method of phylogenetic inference and real value evolutionary trace in order to test the ancestral character of the bifunctional enzyme. Additionally, to search for specificity determinants of these two functions, we have modeled the bifunctional protein and its interactions with both sugar substrates using protein–ligand docking and restricted molecular dynamics. The results show that the evolutionary story of this family is complex. The root of the family is located inside the glucokinase group, showing that the bifunctional enzyme is not an ancestral form, but could be a transitional form from glucokinase to phosphofructokinase, due to its basal location within the phosphofructokinase group. The evolutionary trace and the molecular modeling experiments showed that the specificity for fructose 6‐phosphate is mainly related to the stabilization of a negative charge in the phosphate group, whereas the specificity for glucose is related to the presence of some histidines instead of glutamines/asparagines and to the interaction of this ligand with a glutamic acid residue corresponding to Glu82 in the bifunctional enzyme.

Electron cryo-microscopy structure of the canonical TRPC4 ion channel

Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 Å. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

Catalytic and regulatory roles of divalent metal cations on the phosphoryl-transfer mechanism of ADP-dependent sugar kinases from hyperthermophilic archaea

In some archaea, glucose degradation proceeds through a modified version of the Embden-Meyerhof pathway where glucose and fructose-6-P phosphorylation is carried out by kinases that use ADP as the phosphoryl donor. Unlike their ATP-dependent counterparts these enzymes have been reported as non-regulated. Based on the three dimensional structure determination of several ADP-dependent kinases they can be classified as members of the ribokinase superfamily. In this work, we have studied the role of divalent metal cations on the catalysis and regulation of ADP-dependent glucokinases and phosphofructokinase from hyperthermophilic archaea by means of initial velocity assays as well as molecular dynamics simulations. The results show that a divalent cation is strictly necessary for the activity of these enzymes and they strongly suggest that the true substrate is the metal-nucleotide complex. Also, these enzymes are promiscuous in relation to their metal usage where the only considerations for metal assisted catalysis seem to be related to the ionic radii and coordination geometry of the cations. Molecular dynamics simulations strongly suggest that this metal is bound to the highly conserved NXXE motif, which constitutes one of the signatures of the ribokinase superfamily. Although free ADP cannot act as a phosphoryl donor it still can bind to these enzymes with a reduced affinity, stressing the importance of the metal in the proper binding of the nucleotide at the active site. Also, data show that the binding of a second metal to these enzymes produces a complex with a reduced catalytic constant. On the basis of these findings and considering evolutionary information for the ribokinase superfamily, we propose that the regulatory metal acts by modulating the energy difference between the protein-substrates complex and the reaction transition state, which could constitute a general mechanism for the metal regulation of the enzymes that belong this superfamily.