Franklin A. Hays

A general protocol for the crystallization of membrane proteins for X-ray structural investigation

Protein crystallography is used to generate atomic resolution structures of protein molecules. These structures provide information about biological function, mechanism and interaction of a protein with substrates or effectors including DNA, RNA, cofactors or other small molecules, ions and other proteins. This technique can be applied to membrane proteins resident in the membranes of cells. To accomplish this, membrane proteins first need to be either heterologously expressed or purified from a native source. The protein has to be extracted from the lipid membrane with a mild detergent and purified to a stable, homogeneous population that may then be crystallized. Protein crystals are then used for X-ray diffraction to yield atomic resolution structures of the desired membrane protein target. Below, we present a general protocol for the growth of diffraction quality membrane protein crystals. The process of protein crystallization is highly variable, and obtaining diffraction quality crystals can require weeks to months or even years in some cases.

Selecting Optimum Eukaryotic Integral Membrane Proteins for Structure Determination by Rapid Expression and Solubilization Screening

A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organisms membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-beta-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others.

Caution! DNA Crossing: Crystal Structures of Holliday Junctions

A four-stranded DNA junction (Fig. 1a) was first proposed by Robin Holliday in 1964 as a structural intermediate in a mechanistic model to account for the means by which genetic information is exchanged in yeast (1). This mechanism for genetic exchange is now generally known as homologous recombination and the four-stranded intermediate as the Holliday junction. The general mechanism for recombination has undergone a number of revisions in detail, but the Holliday junction remains a key component in the process and in a growing number of analogous cellular mechanisms (reviewed in Refs. 2 and 3), including site-specific recombination (4), resolution of stalled replication forks (5, 6), DNA repair (7, 8), and phage integration (9). Consequently, the structural and dynamic properties of the Holliday junction have been the focus of intense study since the mid-1960s. As is often the case in science, a multitude of single crystal structures of Holliday junctions in various forms have been solved over a relatively short period of time but only after decades of disappointment. The first structures of junctions in complexes with recombination and DNA repair proteins were reported in 1997 and 1998 (10, 11), whereas junctions in RNA-DNA complexes (12, 13) and in DNA-only constructs (14, 15) emerged just as the twentieth century came to a close. In this review, we will focus on the structures of DNA-only junctions and their geometries, as defined by sequence and ion-dependent interactions.

The stacked-X DNA Holliday junction and protein recognition.

The crystal structure of the four‐stranded DNA Holliday junction has now been determined in the presence and absence of junction binding proteins, with the extended open‐X form of the junction seen in all protein complexes, but the more compact stacked‐X structure observed in free DNA. The structures of the stacked‐X junction were crystallized because of an unexpected sequence dependence on the stability of this structure. Inverted repeat sequences that contain the general motif NCC or ANC favor formation of stacked‐X junctions, with the junction cross‐over occurring between the first two positions of the trinucleotides. This review focuses on the sequence dependent structure of the stacked‐X junction and how it may play a role in structural recognition by a class of dimeric junction resolving enzymes that themselves show no direct sequence recognition. Copyright

BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes

Pro‐apoptotic Bax induces mitochondrial outer membrane permeabilization (MOMP) by forming oligomers through a largely undefined process. Using site‐specific disulfide crosslinking, compartment‐specific chemical labeling, and mutational analysis, we found that activated integral membrane Bax proteins form a BH3‐in‐groove dimer interface on the MOM surface similar to that observed in crystals. However, after the α5 helix was released into the MOM, the remaining interface with α2, α3, and α4 helices was rearranged. Another dimer interface was formed inside the MOM by two intersected or parallel α9 helices. Combinations of these interfaces generated oligomers in the MOM. Oligomerization was initiated by BH3‐in‐groove dimerization, without which neither the other dimerizations nor MOMP occurred. In contrast, α9 dimerization occurred downstream and was required for release of large but not small proteins from mitochondria. Moreover, the release of large proteins was facilitated by α9 insertion into the MOM and localization to the pore rim. Therefore, the BH3‐in‐groove dimerization on the MOM nucleates the assembly of an oligomeric Bax pore that is enlarged by α9 dimerization at the rim.

OVEREXPRESSION AND PURIFICATION OF INTEGRAL MEMBRANE PROTEINS IN YEAST

The budding yeast Saccharomyces cerevisiae is a viable system for the overexpression and functional analysis of eukaryotic integral membrane proteins (IMPs). In this chapter we describe a general protocol for the initial cloning, transformation, overexpression, and subsequent purification of a putative IMP and discuss critical optimization steps and approaches. Since expression and purification are often the two predominant hurdles one will face in studying this difficult class of biological macromolecules the intent is to outline the general workflow while providing insights based upon our collective experience. These insights should facilitate tailoring of the outlined protocol to individual IMPs and expression or purification routines.

A survey of integral α-helical membrane proteins

Membrane proteins serve as cellular gatekeepers, regulators, and sensors. Prior studies have explored the functional breadth and evolution of proteins and families of particular interest, such as the diversity of transport-associated membrane protein families in prokaryotes and eukaryotes, the composition of integral membrane proteins, and family classification of all human G-protein coupled receptors. However, a comprehensive analysis of the content and evolutionary associations between membrane proteins and families in a diverse set of genomes is lacking. Here, a membrane protein annotation pipeline was developed to define the integral membrane genome and associations between 21,379 proteins from 34 genomes; most, but not all of these proteins belong to 598 defined families. The pipeline was used to provide target input for a structural genomics project that successfully cloned, expressed, and purified 61 of our first 96 selected targets in yeast. Furthermore, the methodology was applied (1) to explore the evolutionary history of the substrate-binding transmembrane domains of the human ABC transporter superfamily, (2) to identify the multidrug resistance-associated membrane proteins in whole genomes, and (3) to identify putative new membrane protein families.

FUN26 (function unknown now 26) protein from saccharomyces cerevisiae is a broad selectivity, high affinity, nucleoside and nucleobase transporter

Background: FUN26 is a nucleoside transporter expressed in yeast vacuoles. Results: Proteoliposome studies of purified FUN26 reveal broad nucleoside and nucleobase uptake that is sensitive to C(2′)-ribose modifications. Conclusion: FUN26 is a high affinity and broad selectivity nucleoside and nucleobase transporter. Significance: FUN26 has a unique substrate transport profile relative to other ENTs and retains activity following detergent solubilization and purification. Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2′)- and C(5′)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [3H]Adenine nucleobase transport efficiency is increased ∼4-fold relative to nucleosides tested with no observed [3H]adenosine or [3H]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.

Equilibrative nucleoside transporters-A review

ABSTRACT Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that mediate the transport of nucleosides, nucleobases, and therapeutic analogs. The best-characterized ENTs are the human transporters hENT1 and hENT2. However, non-mammalian eukaryotic ENTs have also been studied (e.g., yeast, parasitic protozoa). ENTs are major pharmaceutical targets responsible for modulating the efficacy of more than 30 approved drugs. However, the molecular mechanisms and chemical determinants of ENT-mediated substrate recognition, binding, inhibition, and transport are poorly understood. This review highlights findings on the characterization of ENTs by surveying studies on genetics, permeant and inhibitor interactions, mutagenesis, and structural models of ENT function.

Imatinib binding to human c-Src is coupled to inter-domain allostery and suggests a novel kinase inhibition strategy

Imatinib (Gleevec), a non-receptor tyrosine kinase inhibitor (nRTKI), is one of the most successful anti-neoplastic drugs in clinical use. However, imatinib-resistant mutations are increasingly prevalent in patient tissues and driving development of novel imatinib analogs. We present a detailed study of the conformational dynamics, in the presence and absence of bound imatinib, for full-length human c-Src using hydrogen-deuterium exchange and mass spectrometry. Our results demonstrate that imatinib binding to the kinase domain effects dynamics of proline-rich or phosphorylated peptide ligand binding sites in distal c-Src SH3 and SH2 domains. These dynamic changes in functional regulatory sites, distal to the imatinib binding pocket, show similarities to structural transitions involved in kinase activation. These data also identify imatinib-sensitive, and imatinib-resistant, mutation sites. Thus, the current study identifies novel c-Src allosteric sites associated with imatinib binding and kinase activation and provide a framework for follow-on development of TKI binding modulators.