RNP-Granule Assembly via Ataxin-2 Disordered Domains Is Required for Long-Term Memory and Neurodegeneration.

Human Ataxin-2 is implicated in the cause and progression of amyotrophic lateral sclerosis (ALS) and type 2 spinocerebellar ataxia (SCA-2). In Drosophila, a conserved atx2 gene is essential for animal survival as well as for normal RNP-granule assembly, translational control, and long-term habituation. Like its human homolog, Drosophila Ataxin-2 (Atx2) contains polyQ repeats and additional intrinsically disordered regions (IDRs). We demonstrate that Atx2 IDRs, which are capable of mediating liquid-liquid phase transitions in vitro, are essential for efficient formation of neuronal mRNP assemblies in vivo. Remarkably, ΔIDR mutants that lack neuronal RNP granules show normal animal development, survival, and fertility. However, they show defects in long-term memory formation/consolidation as well as in C9ORF72 dipeptide repeat or FUS-induced neurodegeneration. Together, our findings demonstrate (1) that higher-order mRNP assemblies contribute to long-term neuronal plasticity and memory, and (2) that a targeted reduction in RNP-granule formation efficiency can alleviate specific forms of neurodegeneration.

Intrinsically Disordered Regions Can Contribute Promiscuous Interactions to RNP Granule Assembly.

Eukaryotic cells contain large RNA-protein assemblies referred to as RNP granules, whose assembly is promoted by both traditional protein interactions and intrinsically disordered protein domains. Using RNP granules as an example, we provide evidence for an assembly mechanism of large cellular structures wherein specific protein-protein or protein-RNA interactions act together with promiscuous interactions of intrinsically disordered regions (IDRs). This synergistic assembly mechanism illuminates RNP granule assembly and explains why many components of RNP granules, and other large dynamic assemblies, contain IDRs linked to specific protein-protein or protein-RNA interaction modules. We suggest assemblies based on combinations of specific interactions and promiscuous IDRs are common features of eukaryotic cells.

Identification of NAD+ capped mRNAs in Saccharomyces cerevisiae.

RNAs besides tRNA and rRNA contain chemical modifications, including the recently described 5' nicotinamide-adenine dinucleotide (NAD+) RNA in bacteria. Whether 5' NAD-RNA exists in eukaryotes remains unknown. We demonstrate that 5' NAD-RNA is found on subsets of nuclear and mitochondrial encoded mRNAs in Saccharomyces cerevisiae NAD-mRNA appears to be produced cotranscriptionally because NAD-RNA is also found on pre-mRNAs, and only on mitochondrial transcripts that are not 5' end processed. These results define an additional 5' RNA cap structure in eukaryotes and raise the possibility that this 5' NAD+ cap could modulate RNA stability and translation on specific subclasses of mRNAs.

A Derived Allosteric Switch Underlies the Evolution of Conditional Cooperativity between HOXA11 and FOXO1.

Transcription factors (TFs) play multiple roles in development. Given this multifunctionality, it has been assumed that TFs are evolutionarily highly constrained. Here, we investigate the molecular mechanisms for the origin of a derived functional interaction between two TFs, HOXA11 and FOXO1. We have previously shown that the regulatory role of HOXA11 in mammalian endometrial stromal cells requires interaction with FOXO1, and that the physical interaction between these proteins evolved before their functional cooperativity. Here, we demonstrate that the derived functional cooperativity between HOXA11 and FOXO1 is due to derived allosteric regulation of HOXA11 by FOXO1. This study shows that TF function can evolve through changes affecting the functional output of a pre-existing protein complex.

Computational design of protein-small molecule interfaces.

The computational design of proteins that bind small molecule ligands is one of the unsolved challenges in protein engineering. It is complicated by the relatively small size of the ligand which limits the number of intermolecular interactions. Furthermore, near-perfect geometries between interacting partners are required to achieve high binding affinities. For apolar, rigid small molecules the interactions are dominated by short-range van der Waals forces. As the number of polar groups in the ligand increases, hydrogen bonds, salt bridges, cation-π, and π-π interactions gain importance. These partial covalent interactions are longer ranged, and additionally, their strength depends on the environment (e.g. solvent exposure). To assess the current state of protein-small molecule interface design, we benchmark the popular computer algorithm Rosetta on a diverse set of 43 protein-ligand complexes. On average, we achieve sequence recoveries in the binding site of 59% when the ligand is allowed limited reorientation, and 48% when the ligand is allowed full reorientation. When simulating the redesign of a protein binding site, sequence recovery among residues that contribute most to binding was 52% when slight ligand reorientation was allowed, and 27% when full ligand reorientation was allowed. As expected, sequence recovery correlates with ligand displacement.

Exploring symmetry as an avenue to the computational design of large protein domains.

It has been demonstrated previously that symmetric, homodimeric proteins are energetically favored, which explains their abundance in nature. It has been proposed that such symmetric homodimers underwent gene duplication and fusion to evolve into protein topologies that have a symmetric arrangement of secondary structure elements--"symmetric superfolds". Here, the ROSETTA protein design software was used to computationally engineer a perfectly symmetric variant of imidazole glycerol phosphate synthase and its corresponding symmetric homodimer. The new protein, termed FLR, adopts the symmetric (ßα)(8) TIM-barrel superfold. The protein is soluble and monomeric and exhibits two-fold symmetry not only in the arrangement of secondary structure elements but also in sequence and at atomic detail, as verified by crystallography. When cut in half, FLR dimerizes readily to form the symmetric homodimer. The successful computational design of FLR demonstrates progress in our understanding of the underlying principles of protein stability and presents an attractive strategy for the in silico construction of larger protein domains from smaller pieces.

Computational design of protein-ligand interfaces: potential in therapeutic development.

Computational design of protein-ligand interfaces finds optimal amino acid sequences within a small-molecule binding site of a protein for tight binding of a specific small molecule. It requires a search algorithm that can rapidly sample the vast sequence and conformational space, and a scoring function that can identify low energy designs. This review focuses on recent advances in computational design methods and their application to protein-small molecule binding sites. Strategies for increasing affinity, altering specificity, creating broad-spectrum binding, and building novel enzymes from scratch are described. Future prospects for applications in drug development are discussed, including limitations that will need to be overcome to achieve computational design of protein therapeutics with novel modes of action.

Computational design of an endo-1,4-beta-xylanase ligand binding site.

The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-ß-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterization of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 ß-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the ß-xylanase proteins.

A physical model for PDZ-domain/peptide interactions.

The PDZ domain is an interaction motif that recognizes and binds the C-terminal peptides of target proteins. PDZ domains are ubiquitous in nature and help assemble multiprotein complexes that control cellular organization and signaling cascades. We present an optimized energy function to predict the binding free energy (ΔΔG) of PDZ domain/peptide interactions computationally. Geometry-optimized models of PDZ domain/peptide interfaces were built using ROSETTA: , and protein and peptide side chain and backbone degrees of freedom are minimized simultaneously. Using leave-one-out cross-validation, ROSETTA: 's energy function is adjusted to reproduce experimentally determined ΔΔG values with a correlation coefficient of 0.66 and a standard deviation of 0.79 kcal mol(-1). The energy function places an increased weight on hydrogen bonding interactions when compared to a previously developed method to analyze protein/protein interactions. Binding free enthalpies (ΔΔH) and entropies (ΔS) are predicted with reduced accuracies of R = 0.60 and R = 0.17, respectively. The computational method improves prediction of PDZ domain specificity from sequence and allows design of novel PDZ domain/peptide interactions.

Human cardiac potassium channel DNA polymorphism modulates access to drug-binding site and causes drug resistance.

Expression of voltage-gated K channel, shaker-related subfamily, member 5 (KCNA5) underlies the human atrial ultra-rapid delayed rectifier K current (I(Kur)). The KCNA5 polymorphism resulting in P532L in the C terminus generates I(Kur) that is indistinguishable from wild type at baseline but strikingly resistant to drug block. In the present study, truncating the C terminus of KCNA5 generated a channel with wild-type drug sensitivity, which indicated that P532 is not a drug-binding site. Secondary structure prediction algorithms identified a probable alpha-helix in P532L that is absent in wild-type channels. We therefore assessed drug sensitivity of I(Kur) generated in vitro in CHO and HEK cells by channels predicted to exhibit or lack this C-terminal alpha-helix. All constructs displayed near-identical I(Kur) in the absence of drug challenge. However, those predicted to lack the C-terminal alpha-helix generated quinidine-sensitive currents (43-51% block by 10 microM quinidine), while the currents generated by those constructs predicted to generate a C-terminal alpha-helix were inhibited less than 12%. Circular dichroism spectroscopy revealed an alpha-helical signature with peptides derived from drug-resistant channels and no organized structure in those associated with wild-type drug sensitivity. In conclusion, we found that this secondary structure in the KCNA5 C terminus, absent in wild-type channels but generated by a naturally occurring DNA polymorphism, does not alter baseline currents but renders the channel drug resistant. Our data support a model in which this structure impairs access of the drug to a pore-binding site.

Calorimetric vs. van't Hoff binding enthalpies from isothermal titration calorimetry: Ba2+-crown ether complexation.

The 1:1 complexation reaction between Ba(2+) and 18-crown-6 ether is re-examined using isothermal titration calorimetry (ITC), with the goal of clarifying previously reported discrepancies between reaction enthalpies estimated directly (calorimetric) and indirectly, from the temperature dependence of the reaction equilibrium constant K (van't Hoff). The ITC thermograms are analyzed using three different non-linear fit models based on different assumptions about the data error: constant, proportional to the heat and proportional but correlated. The statistics of the fitting indicate a preference for the proportional error model, in agreement with expectations for the conditions of the experiment, where uncertainties in the delivered titrant volume should dominate. With attention to proper procedures for propagating statistical error in the van't Hoff analysis, the differences between Delta H(cal) and Delta H(vH) are deemed statistically significant. In addition, statistically significant differences are observed for the Delta H(cal) estimates obtained for two different sources of Ba(2+), BaCl(2) and Ba(NO(3))(2). The effects are tentatively attributed to deficiencies in the standard procedure in ITC of subtracting a blank obtained for pure titrant from the thermogram obtained for the sample.

Designing sequence to control protein function in an EF-hand protein.

The extent of conformational change that calcium binding induces in EF-hand proteins is a key biochemical property specifying Ca(2+) sensor versus signal modulator function. To understand how differences in amino acid sequence lead to differences in the response to Ca(2+) binding, comparative analyses of sequence and structures, combined with model building, were used to develop hypotheses about which amino acid residues control Ca(2+)-induced conformational changes. These results were used to generate a first design of calbindomodulin (CBM-1), a calbindin D(9k) re-engineered with 15 mutations to respond to Ca(2+) binding with a conformational change similar to that of calmodulin. The gene for CBM-1 was synthesized, and the protein was expressed and purified. Remarkably, this protein did not exhibit any non-native-like molten globule properties despite the large number of mutations and the nonconservative nature of some of them. Ca(2+)-induced changes in CD intensity and in the binding of the hydrophobic probe, ANS, implied that CBM-1 does undergo Ca(2+) sensorlike conformational changes. The X-ray crystal structure of Ca(2+)-CBM-1 determined at 1.44 A resolution reveals the anticipated increase in hydrophobic surface area relative to the wild-type protein. A nascent calmodulin-like hydrophobic docking surface was also found, though it is occluded by the inter-EF-hand loop. The results from this first calbindomodulin design are discussed in terms of progress toward understanding the relationships between amino acid sequence, protein structure, and protein function for EF-hand CaBPs, as well as the additional mutations for the next CBM design.

Engineering and design of ligand-induced conformational change in proteins.

The ability to manipulate ligand-induced conformational change, although representing a major challenge to the protein engineer, is an essential end point in efforts to produce novel functional proteins for biotechnology and therapeutic applications. Progress towards this goal requires determining not only what factors control the fold and stability of a protein, but also how ligand binding alters the complex conformational/energetic landscape. Important strides are being made on several fronts, including understanding the origin of long-range effects and allosteric structural mechanisms, using both experimental and theoretical approaches.