A structural basis for the assembly and functions of a viral polymer that inactivates multiple tumor suppressors.

Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central ß core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors.

A Benchmark Study of Protein-Fragment Complex Structure Calculations with NMR2.

Protein-fragment complex structures are particularly sought after in medicinal chemistry to rationally design lead molecules. These structures are usually derived using X-ray crystallography, but the failure rate is non-neglectable. NMR is a possible alternative for the calculation of weakly interacting complexes. Nevertheless, the time-consuming protein signal assignment step remains a barrier to its routine application. NMR Molecular Replacement (NMR2) is a versatile and rapid method that enables the elucidation of a protein-ligand complex structure. It has been successfully applied to peptides, drug-like molecules, and more recently to fragments. Due to the small size of the fragments, ca < 300 Da, solving the structures of the protein-fragment complexes is particularly challenging. Here, we present the expected performances of NMR2 when applied to protein-fragment complexes. The NMR2 approach has been benchmarked with the SERAPhic fragment library to identify the technical challenges in protein-fragment NMR structure calculation. A straightforward strategy is proposed to increase the method's success rate further. The presented work confirms that NMR2 is an alternative method to X-ray crystallography for solving protein-fragment complex structures.

Prebiotic Peptide Synthesis and Spontaneous Amyloid Formation Inside a Proto-Cellular Compartment.

Cellular life requires a high degree of molecular complexity and self-organization, some of which must have originated in a prebiotic context. Here, we demonstrate how both of these features can emerge in a plausibly prebiotic system. We found that chemical gradients in simple mixtures of activated amino acids and fatty acids can lead to the formation of amyloid-like peptide fibrils that are localized inside of a proto-cellular compartment. In this process, the fatty acid or lipid vesicles act both as a filter, allowing the selective passage of activated amino acids, and as a barrier, blocking the diffusion of the amyloidogenic peptides that form spontaneously inside the vesicles. This synergy between two distinct building blocks of life induces a significant increase in molecular complexity and spatial order thereby providing a route for the early molecular evolution that could give rise to a living cell.

Nuclear Magnetic Resonance Solution Structure and Functional Behavior of the Human Proton Channel.

The human voltage-gated proton channel [Hv1(1) or VSDO(2)] plays an important role in the human innate immune system. Its structure differs considerably from those of other cation channels. It is built solely of a voltage-sensing domain and thus lacks the central pore domain, which is essential for other cation channels. Here, we determined the solution structure of an N- and C-terminally truncated human Hv1 (Δ-Hv1) in the resting state by nuclear magnetic resonance (NMR) spectroscopy. Δ-Hv1 comprises the typical voltage-sensing antiparallel four-helix bundle (S1-S4) preceded by an amphipathic helix (S0). The solution structure corresponds to an intermediate state between resting and activated forms of voltage-sensing domains. Furthermore, Zn2+-induced closing of proton channel Δ-Hv1 was studied with two-dimensional NMR spectroscopy, which showed that characteristic large scale dynamics of open Δ-Hv1 are absent in the closed state of the channel. Additionally, pH titration studies demonstrated that a higher H+ concentration is required for the protonation of side chains in the Zn2+-induced closed state than in the open state. These observations demonstrate both structural and dynamical changes involved in the process of voltage gating of the Hv1 channel and, in the future, may help to explain the unique properties of unidirectional conductance and the exceptional ion selectivity of the channel.

Carbonyl Sulfide as a Prebiotic Activation Agent for Stereo- and Sequence-Selective, Amyloid-Templated Peptide Elongation.

Prebiotic chemical replication is a commonly assumed precursor to and prerequisite for life and as such is the one of the goals of our research. We have previously reported on the role that short peptide amyloids could have played in a template-based chemical elongation. Here we take a step closer to the goal by reproducing amyloid-templated peptide elongation with carbonyl sulfide (COS) in place of the less-prebiotically relevant carbonyldiimidazole (CDI) used in the earlier study. Our investigation shows that the sequence-selectivity and stereoselectivity of the amyloid-templated reaction is similar for both activation chemistries. Notably, the amyloid protects the peptides from some of the side-reactions that take place with the COS-activation.

Cooperative Induction of Ordered Peptide and Fatty Acid Aggregates.

Interactions between biological membranes and disease-associated amyloids are well documented, and their prevalence suggests that an inherent affinity exists between these molecular assemblies. Our interest in the molecular origins of life have led us to investigate the nature of such interactions in the context of their molecular predecessors (i.e., vesicle-forming amphiphiles and small peptides). Under certain conditions, amyloidogenic peptides or fatty acids are each able to form ordered structures on their own; however, we report here on their cooperative assembly into novel, to our knowledge, highly ordered structures. We first examined an amyloidogenic eight-residue peptide, which forms amyloids at pH 11, yet because of its positive electrostatic character remains soluble at a neutral pH. In mixtures with simple fatty acids, this peptide is also able to form novel, to our knowledge, coaggregates at a neutral pH whose structures are sensitive to both the fatty acid concentration and identity. Below the critical vesicle concentration, the mixtures of fatty acid and peptide yield a flocculent precipitate with an underlying ß-structure. Above the critical vesicle concentration, the mixtures yield a translucent precipitate that consists of tube-like structures. Small-angle x-ray scattering and fiber diffraction data were used to model their structures as hollow-core two-shell cylinders in which the inner shell is a bilayer of fatty acid and the outer shell alternates between amyloid and bilayers of fatty acid. The further analysis of decanoic acid with a panel of 13 other basic amyloidogenic peptides confirmed the general nature of the observed interactions. The cooperativity within this heterogeneous system is attributed to the structurally repetitive natures of the fatty acid bilayer and the cross-ß-sheet motif, providing compatible scaffolds for attractive electrostatic interactions. We show these interactions to be mutually beneficial, expanding the phase space of both peptides and fatty acids while providing a simple yet robust physical connection between two distinct entities relevant for life.

The mechanism of prion inhibition by HET-S.

HET-S (97% identical to HET-s) has an N-terminal globular domain that exerts a prion-inhibitory effect in cis on its own prion-forming domain (PFD) and in trans on HET-s prion propagation. We show that HET-S fails to form fibrils in vitro and that it inhibits HET-s PFD fibrillization in trans. In vivo analyses indicate that beta-structuring of the HET-S PFD is required for HET-S activity. The crystal structures of the globular domains of HET-s and HET-S are highly similar, comprising a helical fold, while NMR-based characterizations revealed no differences in the conformations of the PFDs. We conclude that prion inhibition is not encoded by structure but rather in stability and oligomerization properties: when HET-S forms a prion seed or is incorporated into a HET-s fibril via its PFD, the beta-structuring in this domain induces a change in its globular domain, generating a molecular species that is incompetent for fibril growth.

Designer nodal/BMP2 chimeras mimic nodal signaling, promote chondrogenesis, and reveal a BMP2-like structure.

Nodal, a member of the TGF-ß superfamily, plays an important role in vertebrate and invertebrate early development. The biochemical study of Nodal and its signaling pathway has been a challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2 library). Three chimeras, named NB250, NB260, and NB264, show Nodal-like signaling properties including dependence on the co-receptor Cripto and activation of the Smad2 pathway. NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and NB260, as well as Nodal, induce chondrogenic differentiation of human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows a backbone scaffold similar to that of BMP2. Our results show that these chimeric ligands may have therapeutic implications in cartilage injuries.

Facile backbone structure determination of human membrane proteins by NMR spectroscopy.

Although nearly half of today's major pharmaceutical drugs target human integral membrane proteins (hIMPs), only 30 hIMP structures are currently available in the Protein Data Bank, largely owing to inefficiencies in protein production. Here we describe a strategy for the rapid structure determination of hIMPs, using solution NMR spectroscopy with systematically labeled proteins produced via cell-free expression. We report new backbone structures of six hIMPs, solved in only 18 months from 15 initial targets. Application of our protocols to an additional 135 hIMPs with molecular weight <30 kDa yielded 38 hIMPs suitable for structural characterization by solution NMR spectroscopy without additional optimization.

An Activin A/BMP2 chimera, AB215, blocks estrogen signaling via induction of ID proteins in breast cancer cells.

BACKGROUND: One in eight women will be affected by breast cancer in her lifetime. Approximately 75% of breast cancers express estrogen receptor alpha (ERα) and/or progesterone receptor and these receptors are markers for tumor dependence on estrogen. Anti-estrogenic drugs such as tamoxifen are commonly used to block estrogen-mediated signaling in breast cancer. However, many patients either do not respond to these therapies (de novo resistance) or develop resistance to them following prolonged treatment (acquired resistance). Therefore, it is imperative to continue efforts aimed at developing new efficient and safe methods of targeting ER activity in breast cancer. METHODS: AB215 is a chimeric ligand assembled from sections of Activin A and BMP2. BMP2's and AB215's inhibition of breast cancer cells growth was investigated. In vitro luciferase and MTT proliferation assays together with western blot, RT_PCR, and mRNA knockdown methods were used to determine the mechanism of inhibition of estrogen positive breast cancer cells growth by BMP2 and AB215. Additionally in vivo xenograft tumor model was used to investigate anticancer properties of AB215. RESULTS: Here we report that AB215, a chimeric ligand assembled from sections of Activin A and BMP2 with BMP2-like signaling, possesses stronger anti-proliferative effects on ERα positive breast cancer cells than BMP2. We further show that AB215 inhibits estrogen signaling by inducing expression of inhibitor of DNA binding proteins (IDs). Specifically, we demonstrate that knockdown of ID proteins attenuates the anti-estrogen effects of AB215. Remarkably, we find that AB215 is more effective than tamoxifen in suppressing tumor growth in a xenograft model. CONCLUSION: This study shows that IDs have profound role to inhibit estrogen signaling in ERα positive breast cancer cells, and that engineered TGF-beta ligands may have high therapeutic value.

Mechanism underlying selective regulation of G protein-gated inwardly rectifying potassium channels by the psychostimulant-sensitive sorting nexin 27.

G protein-gated inwardly rectifying potassium (GIRK) channels are important gatekeepers of neuronal excitability. The surface expression of neuronal GIRK channels is regulated by the psychostimulant-sensitive sorting nexin 27 (SNX27) protein through a class I (-X-Ser/Thr-X-Φ, where X is any residue and Φ is a hydrophobic amino acid) PDZ-binding interaction. The G protein-insensitive inward rectifier channel (IRK1) contains the same class I PDZ-binding motif but associates with a different synaptic PDZ protein, postsynaptic density protein 95 (PSD95). The mechanism by which SNX27 and PSD95 discriminate these channels was previously unclear. Using high-resolution structures coupled with biochemical and functional analyses, we identified key amino acids upstream of the channel's canonical PDZ-binding motif that associate electrostatically with a unique structural pocket in the SNX27-PDZ domain. Changing specific charged residues in the channel's carboxyl terminus or in the PDZ domain converts the selective association and functional regulation by SNX27. Elucidation of this unique interaction site between ion channels and PDZ-containing proteins could provide a therapeutic target for treating brain diseases.

Short Peptide Amyloids Are a Potential Sequence Pool for the Emergence of Proteins.

Under prebiotic conditions, peptides are capable of self-replication through a structure-based template-assisted mechanism when they form amyloids. Furthermore, peptide amyloids can spontaneously form inside fatty acid vesicles creating membrane enclosed complex structures of variable morphologies. This is possible because fatty acid vesicle membranes act as filters allowing passage of activated amino acids while some amino acids derived from the activated species become non-permeable and trapped in the vesicles. Similarly, nascent peptides derived from the condensation of the activated amino acids are also trapped in the vesicles. It is hypothesized that such preselected peptide amyloids become a sequence pool for the emergence of proteins in life and that after billions of years of cellular evolution, the sequences in the current proteome have diverged significantly from these original seed peptides. If this hypothesis is correct, it could be possible to detect the traces of these seed sequences in current proteomes. Here, we show for all possible 3, 6, 7, 8 or 9 residue sequence motifs that those motifs that are most amyloidogenic/aggregation prone are over-represented in extant proteomes compared to a sequence-randomized proteome. Furthermore, we find that there is a greater proportion of amyloidogenic sequence motifs in archaea proteomes than in the larger primate proteomes. This suggests that the evolution towards larger proteomes leads to smaller proportion of amyloidogenic sequences.

On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation.

The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the α-synuclein fibrils hampers efforts in disease-relevant in vitro studies on α-synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of α-synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4, a pH-dependent selection between Type 1, 2, and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro.

Membrane domain structures of three classes of histidine kinase receptors by cell-free expression and rapid NMR analysis.

NMR structural studies of membrane proteins (MP) are hampered by complications in MP expression, technical difficulties associated with the slow process of NMR spectral peak assignment, and limited distance information obtainable for transmembrane (TM) helices. To overcome the inherent challenges in the determination of MP structures, we have developed a rapid and cost-efficient strategy that combines cell-free (CF) protein synthesis, optimized combinatorial dual-isotope labeling for nearly instant resonance assignment, and fast acquisition of long-distance information using paramagnetic probes. Here we report three backbone structures for the TM domains of the three classes of Escherichia coli histidine kinase receptors (HKRs). The ArcB and QseC TM domains are both two-helical motifs, whereas the KdpD TM domain comprises a four-helical bundle with shorter second and third helices. The interhelical distances (up to 12 A) reveal weak interactions within the TM domains of all three receptors. Determined consecutively within 8 months, these structures offer insight into the abundant and underrepresented in the Protein Data Bank class of 2-4 TM crossers and demonstrate the efficiency of our CF combinatorial dual-labeling strategy, which can be applied to solve MP structures in high numbers and at a high speed. Our results greatly expand the current knowledge of HKR structure, opening the doors to studies on their widespread and pharmaceutically important bacterial signaling mechanism.

Separation of domain contacts is required for heterotetrameric assembly of functional NMDA receptors.

The precise knowledge of the subunit assembly process of NMDA receptors (NMDA-Rs) is essential to understand the receptor architecture and underlying mechanism of channel function. Because NMDA-Rs are obligatory heterotetramers requiring the GluN1 subunit, it is critical to investigate how GluN1 and GluN2 type subunits coassemble into tetramers. By combining approaches in cell biology, biochemistry, single particle electron microscopy, and x-ray crystallography, we report the mechanisms and phenotypes of mutant GluN1 subunits that are defective in receptor maturation. The T110A mutation in the N-terminal domain (NTD) of the GluN1 promotes heterodimerization between the NTDs of GluN1 and GluN2, whereas the Y109C mutation in the adjacent residue stabilizes the homodimer of the NTD of GluN1. The crystal structure of the NTD of GluN1 revealed the mechanism underlying the biochemical properties of these mutants. Effects of these mutations on the maturation of heteromeric NMDA-Rs were investigated using a receptor trafficking assay. Our results suggest that the NTDs of the GluN1 subunit initially form homodimers and the subsequent dimer dissociation is critical for forming heterotetrameric NMDA-Rs containing GluN2 subunits, defining a molecular determinant for receptor assembly. The domain arrangement of the dimeric NTD of GluN1 is unique among the ionotropic glutamate receptors and predicts that the structure and mechanism around the NTDs of NMDA-Rs are different from those of the homologous AMPA and kainate receptors.

The functionally active Mistic-fused histidine kinase receptor, EnvZ.

Mistic is a small Bacillus subtilis protein which is of current interest to the field of structural biology and biochemistry because of its unique ability to increase integral membrane protein yields in Escherichia coli expression. Using the osmosensing histidine kinase receptor, EnvZ, an E. coli two-component system, and its cytoplasmic cognate response regulator, OmpR, we provide the first evidence that a Mistic-fused integral membrane protein maintains functionality both in vitro and in vivo. When the purified and detergent-solubilized receptor EnvZ is fused to Mistic, it maintains the ability to autophosphorylate on residue His(243) and phosphotransfers to residue Asp(55) located on OmpR. Functionality was also observed in vivo by means of a ß-galactosidase assay in which RU1012 [Φ(ompC-lacZ)10-15, ΔenvZ::Km(r)] cells transformed with Mistic-fused EnvZ led to an increase in downstream signal transduction events detected by the activation of ompC gene expression. These findings illustrate that Mistic preserves the functionality of the Mistic-fused cargo protein and thus provides a beneficial alternate approach to study integral membrane proteins not only by improving expression levels but also for direct use in functional characterization.

Characterization of protein detergent complexes by NMR, light scattering, and analytical ultracentrifugation.

Bottlenecks in expression, solubilization, purification and crystallization hamper the structural study of integral membrane proteins (IMPs). Successful crystallization is critically dependent on the purity, stability and oligomeric homogeneity of an IMP sample. These characteristics are in turn strongly influenced by the type and concentration of the detergents used in IMP preparation. By utilizing the techniques and analytical tools we earlier developed for the characterization of protein-detergent complexes (PDCs) [21], we demonstrate that for successful protein extraction from E. coli membrane fractions, the solubilizing detergent associates preferentially to IMPs rather than to membrane lipids. Notably, this result is contrary to the generally accepted mechanism of detergent-mediated IMP solubilization. We find that for one particular member of the family of proteins studied (E. coli receptor kinases, which is purified in mixed multimeric states and oligomerizes through its transmembrane region), the protein oligomeric composition is largely unaffected by a 10-fold increase in protein concentration, by alteration of micelle properties through addition of other detergents to the PDC sample, or by a 20-fold variation in the detergent concentration used for solubilization of the IMP from the membrane. We observed that the conditions used for expression of the IMP, which impact protein density in the membrane, has the greatest influence on the IMP oligomeric structure. Finally, we argue that for concentrating PDCs smaller than 30 kDa, stirred concentration cells are less prone to over-concentration of detergent and are therefore more effective than centrifugal ultrafiltration devices.

2007 annual progress report synopsis of the Center for Structures of Membrane Proteins.

A synopsis of the 2007 annual progress report for the Center for Structures of Membrane Proteins, a specialized center of the Protein Structure Initiative.

Cytoplasmic domain structures of Kir2.1 and Kir3.1 show sites for modulating gating and rectification.

N- and C-terminal cytoplasmic domains of inwardly rectifying K (Kir) channels control the ion-permeation pathway through diverse interactions with small molecules and protein ligands in the cytoplasm. Two new crystal structures of the cytoplasmic domains of Kir2.1 (Kir2.1(L)) and the G protein-sensitive Kir3.1 (Kir3.1(S)) channels in the absence of PIP(2) show the cytoplasmic ion-permeation pathways occluded by four cytoplasmic loops that form a girdle around the central pore (G-loop). Significant flexibility of the pore-facing G-loop of Kir2.1(L) and Kir3.1(S) suggests a possible role as a diffusion barrier between cytoplasmic and transmembrane pores. Consistent with this, mutations of the G-loop disrupted gating or inward rectification. Structural comparison shows a di-aspartate cluster on the distal end of the cytoplasmic pore of Kir2.1(L) that is important for modulating inward rectification. Taken together, these results suggest the cytoplasmic domains of Kir channels undergo structural changes to modulate gating and inward rectification.

Peptide Amyloids in the Origin of Life.

How life can emerge from non-living matter is one of the fundamental mysteries of the universe. A bottom-up approach to this problem focuses on the potential chemical precursors of life, in particular the nature of the first replicative molecules. Such thinking has led to the currently most popular idea: that an RNA-like molecule played a central role as the first replicative and catalytic molecule. Here, we review an alternative hypothesis that has recently gained experimental support, focusing on the role of amyloidogenic peptides rather than nucleic acids, in what has been by some termed "the amyloid-world" hypothesis. Amyloids are well-ordered peptide aggregates that have a fibrillar morphology due to their underlying structure of a one-dimensional crystal-like array of peptides in a ß-strand conformation. While they are notorious for their implication in several neurodegenerative diseases including Alzheimer's disease, amyloids also have many biological functions. In this review, we will elaborate on the following properties of amyloids in relation to their fitness as a prebiotic entity: they can be formed by very short peptides with simple amino acids sequences; as aggregates they are more chemically stable than their isolated component peptides; they can possess diverse catalytic activities; they can form spontaneously during the prebiotic condensation of amino acids; they can act as templates in their own chemical replication; they have a structurally repetitive nature that enables them to interact with other structurally repetitive biopolymers like RNA/DNA and polysaccharides, as well as with structurally repetitive surfaces like amphiphilic membranes and minerals.