Elizabeth H. Kellogg

High Resolution Mapping of Protein Sequence–Function Relationships

We present a large-scale approach to investigate the functional consequences of sequence variation in a protein. The approach entails the display of hundreds of thousands of protein variants, moderate selection for activity and high-throughput DNA sequencing to quantify the performance of each variant. Using this strategy, we tracked the performance of >600,000 variants of a human WW domain after three and six rounds of selection by phage display for binding to its peptide ligand. Binding properties of these variants defined a high-resolution map of mutational preference across the WW domain; each position had unique features that could not be captured by a few representative mutations. Our approach could be applied to many in vitro or in vivo protein assays, providing a general means for understanding how protein function relates to sequence.

Structure prediction for CASP8 with all-atom refinement using Rosetta

We describe predictions made using the Rosetta structure prediction methodology for the Eighth Critical Assessment of Techniques for Protein Structure Prediction. Aggressive sampling and all‐atom refinement were carried out for nearly all targets. A combination of alignment methodologies was used to generate starting models from a range of templates, and the models were then subjected to Rosetta all atom refinement. For the 64 domains with readily identified templates, the best submitted model was better than the best alignment to the best template in the Protein Data Bank for 24 cases, and improved over the best starting model for 43 cases. For 13 targets where only very distant sequence relationships to proteins of known structure were detected, models were generated using the Rosetta de novo structure prediction methodology followed by all‐atom refinement; in several cases the submitted models were better than those based on the available templates. Of the 12 refinement challenges, the best submitted model improved on the starting model in seven cases. These improvements over the starting template‐based models and refinement tests demonstrate the power of Rosetta structure refinement in improving model accuracy. Proteins 2009.

High resolution microtubule structures reveal the structural transitions in αβ–tubulin upon GTP hydrolysis

Dynamic instability, the stochastic switching between growth and shrinkage, is essential for microtubule function. This behavior is driven by GTP hydrolysis in the microtubule lattice and is inhibited by anticancer agents like Taxol. We provide insight into the mechanism of dynamic instability, based on high-resolution cryo-EM structures (4.7-5.6 Å) of dynamic microtubules and microtubules stabilized by GMPCPP or Taxol. We infer that hydrolysis leads to a compaction around the E-site nucleotide at longitudinal interfaces, as well as movement of the α-tubulin intermediate domain and H7 helix. Displacement of the C-terminal helices in both α- and β-tubulin subunits suggests an effect on interactions with binding partners that contact this region. Taxol inhibits most of these conformational changes, allosterically inducing a GMPCPP-like state. Lateral interactions are similar in all conditions we examined, suggesting that microtubule lattice stability is primarily modulated at longitudinal interfaces.

Role of conformational sampling in computing mutation‐induced changes in protein structure and stability

The prediction of changes in protein stability and structure resulting from single amino acid substitutions is both a fundamental test of macromolecular modeling methodology and an important current problem as high throughput sequencing reveals sequence polymorphisms at an increasing rate. In principle, given the structure of a wild‐type protein and a point mutation whose effects are to be predicted, an accurate method should recapitulate both the structural changes and the change in the folding‐free energy. Here, we explore the performance of protocols which sample an increasing diversity of conformations. We find that surprisingly similar performances in predicting changes in stability are achieved using protocols that involve very different amounts of conformational sampling, provided that the resolution of the force field is matched to the resolution of the sampling method. Methods involving backbone sampling can in some cases closely recapitulate the structural changes accompanying mutations but not surprisingly tend to do more harm than good in cases where structural changes are negligible. Analysis of the outliers in the stability change calculations suggests areas needing particular improvement; these include the balance between desolvation and the formation of favorable buried polar interactions, and unfolded state modeling. Proteins 2011.

Scientific benchmarks for guiding macromolecular energy function improvement

Accurate energy functions are critical to macromolecular modeling and design. We describe new tools for identifying inaccuracies in energy functions and guiding their improvement, and illustrate the application of these tools to the improvement of the Rosetta energy function. The feature analysis tool identifies discrepancies between structures deposited in the PDB and low-energy structures generated by Rosetta; these likely arise from inaccuracies in the energy function. The optE tool optimizes the weights on the different components of the energy function by maximizing the recapitulation of a wide range of experimental observations. We use the tools to examine three proposed modifications to the Rosetta energy function: improving the unfolded state energy model (reference energies), using bicubic spline interpolation to generate knowledge-based torisonal potentials, and incorporating the recently developed Dunbrack 2010 rotamer library (Shapovalov & Dunbrack, 2011).

Structural differences between yeast and mammalian microtubules revealed by cryo-EM.

Microtubules are polymers of &agr;&bgr;-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between &agr;&bgr;-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. Our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.

Near-atomic cryo-EM structure of PRC1 bound to the microtubule

Significance PRC1 (protein regulator of cytokinesis 1) is critical to cellular architecture through its interaction with microtubules to form antiparallel microtubule arrays, like those in the spindle midzone. Here, cryo-EM studies describe, in close to atomic detail, the interaction of PRC1 with the microtubule surface. Together with previous studies, our structure leads to a model of how PRC1 promotes the establishment of stable, higher-order microtubule arrays. Proteins that associate with microtubules (MTs) are crucial to generate MT arrays and establish different cellular architectures. One example is PRC1 (protein regulator of cytokinesis 1), which cross-links antiparallel MTs and is essential for the completion of mitosis and cytokinesis. Here we describe a 4-Å–resolution cryo-EM structure of monomeric PRC1 bound to MTs. Residues in the spectrin domain of PRC1 contacting the MT are highly conserved and interact with the same pocket recognized by kinesin. We additionally found that PRC1 promotes MT assembly even in the presence of the MT stabilizer taxol. Interestingly, the angle of the spectrin domain on the MT surface corresponds to the previously observed cross-bridge angle between MTs cross-linked by full-length, dimeric PRC1. This finding, together with molecular dynamic simulations describing the intrinsic flexibility of PRC1, suggests that the MT–spectrin domain interface determines the geometry of the MT arrays cross-linked by PRC1.

Arabidopsis thaliana PGR7 Encodes a Conserved Chloroplast Protein That Is Necessary for Efficient Photosynthetic Electron Transport

A significant fraction of a plants nuclear genome encodes chloroplast-targeted proteins, many of which are devoted to the assembly and function of the photosynthetic apparatus. Using digital video imaging of chlorophyll fluorescence, we isolated proton gradient regulation 7 (pgr7) as an Arabidopsis thaliana mutant with low nonphotochemical quenching of chlorophyll fluorescence (NPQ). In pgr7, the xanthophyll cycle and the PSBS gene product, previously identified NPQ factors, were still functional, but the efficiency of photosynthetic electron transport was lower than in the wild type. The pgr7 mutant was also smaller in size and had lower chlorophyll content than the wild type in optimal growth conditions. Positional cloning located the pgr7 mutation in the At3g21200 (PGR7) gene, which was predicted to encode a chloroplast protein of unknown function. Chloroplast targeting of PGR7 was confirmed by transient expression of a GFP fusion protein and by stable expression and subcellular localization of an epitope-tagged version of PGR7. Bioinformatic analyses revealed that the PGR7 protein has two domains that are conserved in plants, algae, and bacteria, and the N-terminal domain is predicted to bind a cofactor such as FMN. Thus, we identified PGR7 as a novel, conserved nuclear gene that is necessary for efficient photosynthetic electron transport in chloroplasts of Arabidopsis.

Insights from the crystal structure of the sixth BRCT domain of topoisomerase IIβ binding protein 1

Topoisomerase IIβ binding protein 1 (TopBP1) is a major player in the DNA damage response and interacts with a number of protein partners via its eight BRCA1 carboxy‐terminal (BRCT) domains. In particular, the sixth BRCT domain of TopBP1 has been implicated in binding to the phosphorylated transcription factor, E2F1, and poly(ADP‐ribose) polymerase 1 (PARP‐1), where the latter interaction is responsible for the poly(ADP‐ribosyl)ation of TopBP1. To gain a better understanding of the nature of TopBP1 BRCT6 interactions, we solved the crystal structure of BRCT6 to 1.34 Å. The crystal structure reveals a degenerate phospho‐peptide binding pocket and lacks conserved hydrophobic residues involved in packing of tandem BRCT repeats, which, together with results from phospho‐peptide binding studies, strongly suggest that TopBP1 BRCT6 independently does not function as a phospho‐peptide binding domain. We further provide insight into poly(ADP‐ribose) binding and sites of potential modification by PARP‐1.

Near-atomic model of microtubule-tau interactions.

Tackling microtubule-tau interactions Alzheimers disease is a major cause of death in the elderly. Disease progression is associated with the accumulation of neurofibrillary tangles composed of tau, a protein important for neuronal development and function. Tangle formation is preceded by phosphorylation events that cause tau to dissociate from its native binding partner, microtubules. Microtubule-tau interactions have been mysterious. Kellogg et al. used cryo–electron microscopy and molecular modeling to show how tau interacts with the outer surface of the microtubule, stapling together tubulin subunits and thus stabilizing the polymer. A key tau amino acid within the tightly bound segment between tubulin subunits corresponds to a clinically relevant site of tau phosphorylation, explaining the competition between microtubule interaction and tau aggregation. Science, this issue p. 1242 A near-atomic model of microtubule-bound tau provides an explanation for disease-associated phosphorylation data. Tau is a developmentally regulated axonal protein that stabilizes and bundles microtubules (MTs). Its hyperphosphorylation is thought to cause detachment from MTs and subsequent aggregation into fibrils implicated in Alzheimer’s disease. It is unclear which tau residues are crucial for tau-MT interactions, where tau binds on MTs, and how it stabilizes them. We used cryo–electron microscopy to visualize different tau constructs on MTs and computational approaches to generate atomic models of tau-tubulin interactions. The conserved tubulin-binding repeats within tau adopt similar extended structures along the crest of the protofilament, stabilizing the interface between tubulin dimers. Our structures explain the effect of phosphorylation on MT affinity and lead to a model of tau repeats binding in tandem along protofilaments, tethering together tubulin dimers and stabilizing polymerization interfaces.