α-Tocopherol transfer protein does not regulate the cellular uptake and intracellular distribution of α- and γ-tocopherols and -tocotrienols in cultured liver cells.

Liver cells express a cytosolic α-tocopherol transfer protein (αTTP) with high binding affinity for α-tocopherol (αT) and much lower affinities for the non-αT congeners. The role of αTTP in the intracellular distribution of the different vitamin E forms is currently unknown. We therefore investigated the intracellular localization of αT, γ-tocopherol (γT), α-tocotrienol (αT3), and γ-tocotrienol (γT3) in cultured hepatic cells with and without stable expression of αTTP. We first determined cellular uptake of the four congeners and found the methylation of the chromanol ring and saturation of the sidechain to be important factors, with tocotrienols being taken up more efficiently than tocopherols and the γ-congeners more than the α-congeners, irrespective of the expression of αTTP. This, however, could perhaps also be due to an observed higher stability of tocotrienols, compared to tocopherols, in culture media rather than a higher absorption. We then incubated HepG2 cells and αTTP-expressing HepG2 cells with αT, γT, αT3, or γT3, isolated organelle fractions by density gradient centrifugation, and determined the concentrations of the congeners in the subcellular fractions. All four congeners were primarily associated with the lysosomes, endoplasmic reticulum, and plasma membrane, whereas only αT correlated with mitochondria. Neither the chromanol ring methylation or sidechain saturation, nor the expression of αTTP were important factors for the intracellular distribution of vitamin E. In conclusion, αTTP does not appear to regulate the uptake and intracellular localization of different vitamin E congeners in cultured liver cells.

Structural reconstruction of protein ancestry.

Ancestral protein reconstruction allows the resurrection and characterization of ancient proteins based on computational analyses of sequences of modern-day proteins. Unfortunately, many protein families are highly divergent and not suitable for sequence-based reconstruction approaches. This limitation is exemplified by the antigen receptors of jawed vertebrates (B- and T-cell receptors), heterodimers formed by pairs of Ig domains. These receptors are believed to have evolved from an extinct homodimeric ancestor through a process of gene duplication and diversification; however molecular evidence has so far remained elusive. Here, we use a structural approach and laboratory evolution to reconstruct such molecules and characterize their interaction with antigen. High-resolution crystal structures of reconstructed homodimeric receptors in complex with hen-egg white lysozyme demonstrate how nanomolar affinity binding of asymmetrical antigen is enabled through selective recruitment and structural plasticity within the receptor-binding site. Our results provide structural evidence in support of long-held theories concerning the evolution of antigen receptors, and provide a blueprint for the experimental reconstruction of protein ancestry in the absence of phylogenetic evidence.

Cryo-EM analysis of a domain antibody bound rotary ATPase complex.

The bacterial A/V-type ATPase/synthase rotary motor couples ATP hydrolysis/synthesis with proton translocation across biological membranes. The A/V-type ATPase/synthase from Thermus thermophilus has been extensively studied both structurally and functionally for many years. Here we provide an 8.7Å resolution cryo-electron microscopy 3D reconstruction of this complex bound to single-domain antibody fragments, small monomeric antibodies containing just the variable heavy domain. Docking of known structures into the density revealed the molecular orientation of the domain antibodies, suggesting that structure determination of co-domain antibody:protein complexes could be a useful avenue for unstable or smaller proteins. Although previous studies suggested that the presence of fluoroaluminate in this complex could change the rotary state of this enzyme, we observed no gross structural rearrangements under these conditions.

Cryo-EM structures of the autoinhibited E. coli ATP synthase in three rotational states.

A molecular model that provides a framework for interpreting the wealth of functional information obtained on the E. coli F-ATP synthase has been generated using cryo-electron microscopy. Three different states that relate to rotation of the enzyme were observed, with the central stalk's ε subunit in an extended autoinhibitory conformation in all three states. The Fo motor comprises of seven transmembrane helices and a decameric c-ring and invaginations on either side of the membrane indicate the entry and exit channels for protons. The proton translocating subunit contains near parallel helices inclined by ~30° to the membrane, a feature now synonymous with rotary ATPases. For the first time in this rotary ATPase subtype, the peripheral stalk is resolved over its entire length of the complex, revealing the F1 attachment points and a coiled-coil that bifurcates toward the membrane with its helices separating to embrace subunit a from two sides.

Structural and Functional Insights into the Evolution and Stress Adaptation of Type II Chaperonins.

Chaperonins are essential biological complexes assisting protein folding in all kingdoms of life. Whereas homooligomeric bacterial GroEL binds hydrophobic substrates non-specifically, the heterooligomeric eukaryotic CCT binds specifically to distinct classes of substrates. Sulfolobales, which survive in a wide range of temperatures, have evolved three different chaperonin subunits (α, ß, γ) that form three distinct complexes tailored for different substrate classes at cold, normal, and elevated temperatures. The larger octadecameric ß complexes cater for substrates under heat stress, whereas smaller hexadecameric αß complexes prevail under normal conditions. The cold-shock complex contains all three subunits, consistent with greater substrate specificity. Structural analysis using crystallography and electron microscopy reveals the geometry of these complexes and shows a novel arrangement of the α and ß subunits in the hexadecamer enabling incorporation of the γ subunit.

Domain-swap polymerization drives the self-assembly of the bacterial flagellar motor.

Large protein complexes assemble spontaneously, yet their subunits do not prematurely form unwanted aggregates. This paradox is epitomized in the bacterial flagellar motor, a sophisticated rotary motor and sensory switch consisting of hundreds of subunits. Here we demonstrate that Escherichia coli FliG, one of the earliest-assembling flagellar motor proteins, forms ordered ring structures via domain-swap polymerization, which in other proteins has been associated with uncontrolled and deleterious protein aggregation. Solution structural data, in combination with in vivo biochemical cross-linking experiments and evolutionary covariance analysis, revealed that FliG exists predominantly as a monomer in solution but only as domain-swapped polymers in assembled flagellar motors. We propose a general structural and thermodynamic model for self-assembly, in which a structural template controls assembly and shapes polymer formation into rings.

Rotary ATPases--dynamic molecular machines.

Recent work has provided the detailed overall architecture and subunit composition of three subtypes of rotary ATPases. Composite models of F-type, V-type and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual components into electron microscopy derived envelopes of the intact enzymes. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria. An inherent flexibility in rotary ATPases observed by different techniques suggests greater dynamics during operation than previously envisioned. The concerted movement of subunits within the complex might provide means of regulation and information transfer between distant parts of rotary ATPases thereby fine tuning these molecular machines to their cellular environment, while optimizing their efficiency.

Ion mobility-mass spectrometry of a rotary ATPase reveals ATP-induced reduction in conformational flexibility.

Rotary ATPases play fundamental roles in energy conversion as their catalytic rotation is associated with interdomain fluctuations and heterogeneity of conformational states. Using ion mobility mass spectrometry we compared the conformational dynamics of the intact ATPase from Thermus thermophilus with those of its membrane and soluble subcomplexes. Our results define regions with enhanced flexibility assigned to distinct subunits within the overall assembly. To provide a structural context for our experimental data we performed molecular dynamics simulations and observed conformational changes of the peripheral stalks that reflect their intrinsic flexibility. By isolating complexes at different phases of cell growth and manipulating nucleotides, metal ions and pH during isolation, we reveal differences that can be related to conformational changes in the Vo complex triggered by ATP binding. Together these results implicate nucleotides in modulating flexibility of the stator components and uncover mechanistic detail that underlies operation and regulation in the context of the holoenzyme.

Purification of molecular machines and nanomotors using phage-derived monoclonal antibody fragments.

Molecular machines and nanomotors are sophisticated biological assemblies that convert potential energy stored either in transmembrane ion gradients or in ATP into kinetic energy. Studying these highly dynamic biological devices by X-ray crystallography is challenging, as they are difficult to produce, purify, and crystallize. Phage display technology allows us to put a handle on these molecules in the form of highly specific antibody fragments that can also stabilize conformations and allow versatile labelling for electron microscopy, immunohistochemistry, and biophysics experiments.Here, we describe a widely applicable protocol for selecting high-affinity monoclonal antibody fragments against a complex molecular machine, the A-type ATPase from T. thermophilus that allows fast and simple purification of this transmembrane rotary motor from its wild-type source. The approach can be readily extended to other integral membrane proteins and protein complexes as well as to soluble molecular machines and nanomotors.

Rotary ATPases: models, machine elements and technical specifications.

Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual "machine elements" to the requirement of the right "fuel" and "oil" for different types of motors.

Priming a molecular motor for disassembly.

In this issue of Structure, Oot and colleagues present the crystal structure of the eukaryotic V-ATPase peripheral stalk in complex with one of its binding partners, revealing conformational flexibility that may be important for priming the complex for rapid disassembly in response to external stimuli.

General strategy for the generation of human antibody variable domains with increased aggregation resistance.

The availability of stable human antibody reagents would be of considerable advantage for research, diagnostic, and therapeutic applications. Unfortunately, antibody variable heavy and light domains (V(H) and V(L)) that mediate the interaction with antigen have the propensity to aggregate. Increasing their aggregation resistance in a general manner has proven to be a difficult and persistent problem, due to the high level of sequence diversity observed in human variable domains and the requirement to maintain antigen binding. Here we outline such an approach. By using phage display we identified specific positions that clustered in the antigen binding site (28, 30-33, 35 in V(H) and 24, 49-53, 56 in V(L)). Introduction of aspartate or glutamate at these positions endowed superior biophysical properties (non-aggregating, well-expressed, and heat-refoldable) onto domains derived from common human germline families (V(H)3 and V(κ)1). The effects of the mutations were highly positional and independent of sequence diversity at other positions. Moreover, crystal structures of mutant V(H) and V(L) domains revealed a surprising degree of structural conservation, indicating compatibility with V(H)/V(L) pairing and antigen binding. This allowed the retrofitting of existing binders, as highlighted by the development of robust high affinity antibody fragments derived from the breast cancer therapeutic Herceptin. Our results provide a general strategy for the generation of human antibody variable domains with increased aggregation resistance.

The dynamic stator stalk of rotary ATPases.

Rotary ATPases couple ATP hydrolysis/synthesis with proton translocation across biological membranes and so are central components of the biological energy conversion machinery. Their peripheral stalks are essential components that counteract torque generated by rotation of the central stalk during ATP synthesis or hydrolysis. Here we present a 2.25-Å resolution crystal structure of the peripheral stalk from Thermus thermophilus A-type ATPase/synthase. We identify bending and twisting motions inherent within the structure that accommodate and complement a radial wobbling of the ATPase headgroup as it progresses through its catalytic cycles, while still retaining azimuthal stiffness necessary to counteract rotation of the central stalk. The conformational freedom of the peripheral stalk is dictated by its unusual right-handed coiled-coil architecture, which is in principle conserved across all rotary ATPases. In context of the intact enzyme, the dynamics of the peripheral stalks provides a potential mechanism for cooperativity between distant parts of rotary ATPases.

Nanorotors and self-assembling macromolecular machines: the torque ring of the bacterial flagellar motor.

The bacterial flagellar motor (BFM) is a self-assembling rotary nanomachine. It converts a flux of cations into the mechanical rotation of long filaments that propel bacteria through viscous media. The BFM contains a torque-generating ring that is complete with molecular machinery known as the switch complex that allows it to reverse directions. With four billion years of optimization, the BFM probably offers the pinnacle of sophisticated nanorotor design. Moreover as one of the best-characterized large biomolecular complexes, it offers the potential for convergence between nanotechnology and biology, which requires an atomic level understanding of BFM structure and function. This review focuses on current molecular models of the reversible BFM and the strategies used to derive them.

Mass spectrometry of intact V-type ATPases reveals bound lipids and the effects of nucleotide binding.

The ability of electrospray to propel large viruses into a mass spectrometer is established and is rationalized by analogy to the atmospheric transmission of the common cold. Much less clear is the fate of membrane-embedded molecular machines in the gas phase. Here we show that rotary adenosine triphosphatases (ATPases)/synthases from Thermus thermophilus and Enterococcus hirae can be maintained intact with membrane and soluble subunit interactions preserved in vacuum. Mass spectra reveal subunit stoichiometries and the identity of tightly bound lipids within the membrane rotors. Moreover, subcomplexes formed in solution and gas phases reveal the regulatory effects of nucleotide binding on both ATP hydrolysis and proton translocation. Consequently, we can link specific lipid and nucleotide binding with distinct regulatory roles.

The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation.

The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS) domain (residues 26-135) as well as an amphipathic α-helix (residues 13-23) and an initial unstructured segment (residues 2-9). Deletion of residues 2-25, only the unstructured segment (residues 2-9) or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.

The C-H peripheral stalk base: a novel component in V1-ATPase assembly.

Vacuolar ATPases (V-ATPases) are molecular machines responsible for creating electrochemical gradients and preserving pH-dependent cellular compartments by way of proton translocation across the membrane. V-ATPases employ a dynamic rotary mechanism that is driven by ATP hydrolysis and the central rotor stalk. Regulation of this rotational catalysis is the result of a reversible V(1)V(o)-domain dissociation that is required to preserve ATP during instances of cellular starvation. Recently the method by which the free V(1)-ATPase abrogates the hydrolytic breakdown of ATP upon dissociating from the membrane has become increasingly clear. In this instance the central stalk subunit F adopts an extended conformation to engage in a bridging interaction tethering the rotor and stator components together. However, the architecture by which this mechanism is stabilized has remained ambiguous despite previous work. In an effort to elucidate the method by which the rotational catalysis is maintained, the architecture of the peripheral stalks and their respective binding interactions was investigated using cryo-electron microscopy. In addition to confirming the bridging interaction exuded by subunit F for the first time in a eukaryotic V-ATPase, subunits C and H are seen interacting with one another in a tight interaction that provides a base for the three EG peripheral stalks. The formation of a CE(3)G(3)H sub-assembly appears to be unique to the dissociated V-ATPase and highlights the stator architecture in addition to revealing a possible intermediate in the assembly mechanism of the free V(1)-ATPase.

Structure of the torque ring of the flagellar motor and the molecular basis for rotational switching.

The flagellar motor drives the rotation of flagellar filaments at hundreds of revolutions per second, efficiently propelling bacteria through viscous media. The motor uses the potential energy from an electrochemical gradient of cations across the cytoplasmic membrane to generate torque. A rapid switch from anticlockwise to clockwise rotation determines whether a bacterium runs smoothly forward or tumbles to change its trajectory. A protein called FliG forms a ring in the rotor of the flagellar motor that is involved in the generation of torque through an interaction with the cation-channel-forming stator subunit MotA. FliG has been suggested to adopt distinct conformations that induce switching but these structural changes and the molecular mechanism of switching are unknown. Here we report the molecular structure of the full-length FliG protein, identify conformational changes that are involved in rotational switching and uncover the structural basis for the formation of the FliG torque ring. This allows us to propose a model of the complete ring and switching mechanism in which conformational changes in FliG reverse the electrostatic charges involved in torque generation.

The structure of the peripheral stalk of Thermus thermophilus H+-ATPase/synthase.

Proton-translocating ATPases are ubiquitous protein complexes that couple ATP catalysis with proton translocation via a rotary catalytic mechanism. The peripheral stalks are essential components that counteract torque generated from proton translocation during ATP synthesis or from ATP hydrolysis during proton pumping. Despite their essential role, the peripheral stalks are the least conserved component of the complexes, differing substantially between subtypes in composition and stoichiometry. We have determined the crystal structure of the peripheral stalk of the A-type ATPase/synthase from Thermus thermophilus consisting of subunits E and G. The structure contains a heterodimeric right-handed coiled coil, a protein fold never observed before. We have fitted this structure into the 23 A resolution EM density of the intact A-ATPase complex, revealing the precise location of the peripheral stalk and new implications for the function and assembly of proton-translocating ATPases.

Stoichiometry and localization of the stator subunits E and G in Thermus thermophilus H+-ATPase/synthase.

Proton-translocating ATPases are central to biological energy conversion. Although eukaryotes contain specialized F-ATPases for ATP synthesis and V-ATPases for proton pumping, eubacteria and archaea typically contain only one enzyme for both tasks. Although many eubacteria contain ATPases of the F-type, some eubacteria and all known archaea contain ATPases of the A-type. A-ATPases are closely related to V-ATPases but simpler in design. Although the nucleotide-binding and transmembrane rotor subunits share sequence homology between A-, V-, and F-ATPases, the peripheral stalk is strikingly different in sequence, composition, and stoichiometry. We have analyzed the peripheral stalk of Thermus thermophilus A-ATPase by using phage display-derived single-domain antibody fragments in combination with electron microscopy and tandem mass spectrometry. Our data provide the first direct evidence for the existence of two peripheral stalks in the A-ATPase, each one composed of heterodimers of subunits E and G arranged symmetrically around the soluble A(1) domain. To our knowledge, this is the first description of phage display-derived antibody selection against a multi-subunit membrane protein used for purification and single particle analysis by electron microscopy. It is also the first instance of the derivation of subunit stoichiometry by tandem mass spectrometry to an intact membrane protein complex. Both approaches could be applicable to the structural analysis of other membrane protein complexes.