Jacqueline M. Gulbis

Domain Reorientation and Rotation of an Intracellular Assembly Regulate Conduction in Kir Potassium Channels.

Potassium channels embedded in cell membranes employ gates to regulate K+ current. While a specific constriction in the permeation pathway has historically been implicated in gating, recent reports suggest that the signature ion selectivity filter located in the outer membrane leaflet may be equally important. Inwardly rectifying K+ channels also control the directionality of flow, using intracellular polyamines to stem ion efflux by a valve-like action. This study presents crystallographic evidence of interdependent gates in the conduction pathway and reveals the mechanism of polyamine block. Reorientation of the intracellular domains, concomitant with activation, instigates polyamine release from intracellular binding sites to block the permeation pathway. Conformational adjustments of the slide helices, achieved by rotation of the cytoplasmic assembly relative to the pore, are directly correlated to the ion configuration in the selectivity filter. Ion redistribution occurs irrespective of the constriction, suggesting a more expansive role of the selectivity filter in gating than previously appreciated.

Structural and Functional Requirements for Activity of the Tim9–Tim10 Complex in Mitochondrial Protein Import

The Tim9-Tim10 complex plays an essential role in mitochondrial protein import by chaperoning select hydrophobic precursor proteins across the intermembrane space. How the complex interacts with precursors is not clear, although it has been proposed that Tim10 acts in substrate recognition, whereas Tim9 acts in complex stabilization. In this study, we report the structure of the yeast Tim9-Tim10 hexameric assembly determined to 2.5 A and have performed mutational analysis in yeast to evaluate the specific roles of Tim9 and Tim10. Like the human counterparts, each Tim9 and Tim10 subunit contains a central loop flanked by disulfide bonds that separate two extended N- and C-terminal tentacle-like helices. Buried salt-bridges between highly conserved lysine and glutamate residues connect alternating subunits. Mutation of these residues destabilizes the complex, causes defective import of precursor substrates, and results in yeast growth defects. Truncation analysis revealed that in the absence of the N-terminal region of Tim9, the hexameric complex is no longer able to efficiently trap incoming substrates even though contacts with Tim10 are still made. We conclude that Tim9 plays an important functional role that includes facilitating the initial steps in translocating precursor substrates into the intermembrane space.

Translocation of a Bak C-Terminus Mutant from Cytosol to Mitochondria to Mediate Cytochrome c Release: Implications for Bak and Bax Apoptotic Function

Background One of two proapoptotic Bcl-2 proteins, Bak or Bax, is required to permeabilize the mitochondrial outer membrane during apoptosis. While Bax is mostly cytosolic and translocates to mitochondria following an apoptotic stimulus, Bak is constitutively integrated within the outer membrane. Membrane anchorage occurs via a C-terminal transmembrane domain that has been studied in Bax but not in Bak, therefore what governs their distinct subcellular distribution is uncertain. In addition, whether the distinct subcellular distributions of Bak and Bax contributes to their differential regulation during apoptosis remains unclear. Methodology/Principal Findings To gain insight into Bak and Bax targeting to mitochondria, elements of the Bak C-terminus were mutated, or swapped with those of Bax. Truncation of the C-terminal six residues (C-segment) or substitution of three basic residues within the C-segment destabilized Bak. Replacing the Bak C-segment with that from Bax rescued stability and function, but unexpectedly resulted in a semi-cytosolic protein, termed Bak/BaxCS. When in the cytosol, both Bax and Bak/BaxCS sequestered their hydrophobic transmembrane domains in their hydrophobic surface groove. Upon apoptotic signalling, Bak/BaxCS translocated to the mitochondrial outer membrane, inserted its transmembrane domain, oligomerized, and released cytochrome c. Despite this Bax-like subcellular distribution, Bak/BaxCS retained Bak-like regulation following targeting of Mcl-1. Conclusions/Significance Residues in the C-segment of Bak and of Bax contribute to their distinct subcellular localizations. That a semi-cytosolic form of Bak, Bak/BaxCS, could translocate to mitochondria and release cytochrome c indicates that Bak and Bax share a conserved mode of activation. In addition, the differential regulation of Bak and Bax by Mcl-1 is predominantly independent of the initial subcellular localizations of Bak and Bax.

Minimal requirements for actin filament disassembly revealed by structural analysis of malaria parasite actin-depolymerizing factor 1

Malaria parasite cell motility is a process that is dependent on the dynamic turnover of parasite-derived actin filaments. Despite its central role, actins polymerization state is controlled by a set of identifiable regulators that is markedly reduced compared with those of other eukaryotic cells. In Plasmodium falciparum, the most virulent species that affects humans, this minimal repertoire includes two members of the actin-depolymerizing factor/cofilin (AC) family of proteins, P. falciparum actin-depolymerizing factor 1 (PfADF1) and P. falciparum actin-depolymerizing factor 2. This essential class of actin regulator is involved in the control of filament dynamics at multiple levels, from monomer binding through to filament depolymerization and severing. Previous biochemical analyses have suggested that PfADF1 sequesters monomeric actin but, unlike most eukaryotic counterparts, has limited potential to bind or depolymerize filaments. The molecular basis for these unusual properties and implications for parasite cell motility have not been established. Here we present the crystal structure of an apicomplexan AC protein, PfADF1. We show that PfADF1 lacks critical residues previously implicated as essential for AC-mediated actin filament binding and disassembly, having a substantially reduced filament-binding loop and C-terminal α4 helix. Despite this divergence in structure, we demonstrate that PfADF1 is capable of efficient actin filament severing. Furthermore, this severing occurs despite PfADF1’s low binding affinity for filaments. Comparative structural analysis along with biochemical and microscopy evidence establishes that severing is reliant on the availability of an exposed basic residue in the filament-binding loop, a conserved minimal requirement that defines AC-mediated filament disassembly across eukaryotic cells.

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.

Oligomerization at the membrane: potassium channel structure and function.

Cell membranes present a naturally impervious barrier to aqueous solutes, such that the physiochemical environment on either side of the lipid bilayer can substantially differ. Integral membrane proteins are embedded in this heterogeneous lipid environment, wherein the juxtaposition of apolar and polar molecular surfaces defines factors such as transverse orientation, the surface area available for oligomerisation and the symmetry of resultant assemblies. This chapter focuses on potassium channels -representative molecular pores that play a critical role in electrical signalling by enabling selective transport of K(+) ions across cell membranes. Oligomerization is central to K(+) channel action; individual subunits are nonfunctional and conduction, selectivity and gating involve manipulation of the common subunit interface of the tetramer. Regulation of channel activity can be viewed from the perspective that the pore of K(+) channels has coopted other proteins, utilizing a process of hetero-oligomerisation to absorb new functions that both enable the pore to respond to extrinsic signals and provide an electrical signature.

Engineering Protocells: Prospects for Self-Assembly and Nanoscale Production-Lines

The increasing ease of producing nucleic acids and proteins to specification offers potential for design and fabrication of artificial synthetic “organisms” with a myriad of possible capabilities. The prospects for these synthetic organisms are significant, with potential applications in diverse fields including synthesis of pharmaceuticals, sources of renewable fuel and environmental cleanup. Until now, artificial cell technology has been largely restricted to the modification and metabolic engineering of living unicellular organisms. This review discusses emerging possibilities for developing synthetic protocell “machines” assembled entirely from individual biological components. We describe a host of recent technological advances that could potentially be harnessed in design and construction of synthetic protocells, some of which have already been utilized toward these ends. More elaborate designs include options for building self-assembling machines by incorporating cellular transport and assembly machinery. We also discuss production in miniature, using microfluidic production lines. While there are still many unknowns in the design, engineering and optimization of protocells, current technologies are now tantalizingly close to the capabilities required to build the first prototype protocells with potential real-world applications.

Structure and function of LGR5: An enigmatic G-protein coupled receptor marking stem cells

G‐protein coupled receptors (GPCRs) are an important class of membrane protein that transmit extracellular signals invoked by sensing molecules such as hormones and neurotransmitters. GPCR dysfunction is implicated in many diseases and hence these proteins are of great interest to academia and the pharmaceutical industry. Leucine‐rich repeat‐containing GPCRs contain a characteristic extracellular domain that is an important modulator of intracellular signaling. One member of this class is the leucine‐rich repeat‐containing G‐protein‐coupled receptor 5 (LGR5), a stem cell marker in intestinal crypts, and mammary glands. LGR5 modulates Wnt signaling in the presence of the ligand R‐spondin (RSPO). The mechanism of activation of LGR5 by RSPO is not understood, nor is the intracellular signaling mechanism known. Recently reported structures of the extracellular domain of LGR5 bound to RSPO reveal a horseshoe‐shaped architecture made up of consecutive leucine‐rich repeats, with RSPO bound on the concave surface. This review discusses the discovery of LGR5 and the impact it is having on our understanding of stem cell and cancer biology of the colon. In addition, it covers functional relationships suggested by sequence homology and structural analyses, as well as some intriguing conundrums with respect to the involvement of LGR5 in Wnt signaling.

Potassium channel gating: Not an open and shut case

Potassium channels are highly complex molecular systems that modulate the cell potential in response to extracellular or intracellular signals. In multicellular organisms, K+ currents are essential to electrical signaling, governing central nervous system, cardiac, renal, and a host of other organ functions. Publication of the first K+ channel structure in 1998 (1) represented a landmark in our understanding of the molecular basis for channel function, revealing for the first time the overall architecture of an ion channel and illuminating the basis of the channels selectivity for K+ over other cations. The subject of that study was the pH-activated prokaryotic channel KcsA. Despite the determination of structures of other channel classes, and extensive supporting biophysical data, the means by which K+ conduction is switched on and off has remained elusive.

Protein crystallography: methods and protocols

[1] Ilavsky J, Jemian PR. Irena: tool suite for modeling and analysis of small-angle scattering. J Appl Crystallogr. 2009;42:347–353. [2] Sakdinawat A, Attwood D. Nanoscale X-ray imaging. Nat Photonics. 2010;4:840–848. [3] Cheng Y, Suhonen H, Helfen L, et al. Direct three-dimensional imaging of polymer–water interfaces by nanoscale hard X-ray phase tomography. Soft Matter. 2014;10:2982–2990. [4] Sorrentino A, Nicolas J, Valcarcel R, et al. MISTRAL: a transmission soft X-ray microscopy beamline for cryo nano-tomography of biological samples and magnetic domains imaging. J Sychrotron Rad. 2015;22(4):1112–1117. [5] Liu YS, Glans PA, Chuang CH, et al. Perspectives of in situ/operando resonant inelastic X-ray scattering in catalytic energy materials science. J Electron Spectrosc Relat Phenom. 2015;200:282–292. [6] Braun A, Nordlund D, Song SW, et al. Hard X-rays in-soft X-rays out: an operando piggyback view deep into a charging lithium ion battery with X-ray Raman spectroscopy. J Electron Spectrosc Relat Phenom. 2015;200:257–263. [7] Cnossen I, Forcada JS, Favata F, et al. Habitat of early life: solar X-ray and UV radiation at Earth’s surface 4–3.5 billion years ago. J Geophys Res: Planets. 2007;112:E02008. DOI:10.1029/2006JE002784