Evolving a Thermostable Terminal Deoxynucleotidyl Transferase.

Terminal deoxynucleotidyl transferase (TdT) catalyzes template free incorporation of arbitrary nucleotides onto single-stranded DNA. Due to this unique feature, TdT is widely used in biotechnology and clinical applications. One particularly tantalizing use is the synthesis of long de novo DNA molecules by TdT-mediated iterative incorporation of a 3' reversibly blocked nucleotide, followed by deblocking. However, wild-type (WT) TdT is not optimized for the incorporation of 3' modified nucleotides, and TdT engineering is hampered by the fact that TdT is marginally stable and only present in mesophilic organisms. We sought to first evolve a thermostable TdT variant to serve as backbone for subsequent evolution to enable efficient incorporation of 3'-modified nucleotides. A thermostable variant would be a good starting point for such an effort, as evolution to incorporate bulky modified nucleotides generally results in lowered stability. In addition, a thermostable TdT would also be useful when blunt dsDNA is a substrate as higher temperature could be used to melt dsDNA. Here, we developed an assay to identify thermostable TdT variants. After screening about 10 000 TdT mutants, we identified a variant, named TdT3-2, that is 10 °C more thermostable than WT TdT, while preserving the catalytic properties of the WT enzyme.

Novel function of a dynein light chain in actin assembly during clathrin-mediated endocytosis.

Clathrin- and actin-mediated endocytosis is essential in eukaryotic cells. In this study, we demonstrate that Tda2 is a novel protein of the endocytic machinery necessary for normal internalization of native cargo in yeast. Tda2 has not been classified in any protein family. Unexpectedly, solving the crystal structure of Tda2 revealed it belongs to the dynein light chain family. However, Tda2 works independently of the dynein motor complex and microtubules. Tda2 forms a tight complex with the polyproline motif-rich protein Aim21, which interacts physically with the SH3 domain of the Arp2/3 complex regulator Bbc1. The Tda2-Aim21 complex localizes to endocytic sites in a Bbc1- and filamentous actin-dependent manner. Importantly, the Tda2-Aim21 complex interacts directly with and facilitates the recruitment of actin-capping protein, revealing barbed-end filament capping at endocytic sites to be a regulated event. Thus, we have uncovered a new layer of regulation of the actin cytoskeleton by a member of a conserved protein family that has not been previously associated with a function in endocytosis.

Design of a Genetically Stable High Fidelity Coxsackievirus B3 Polymerase That Attenuates Virus Growth in Vivo.

Positive strand RNA viruses replicate via a virally encoded RNA-dependent RNA polymerase (RdRP) that uses a unique palm domain active site closure mechanism to establish the canonical two-metal geometry needed for catalysis. This mechanism allows these viruses to evolutionarily fine-tune their replication fidelity to create an appropriate distribution of genetic variants known as a quasispecies. Prior work has shown that mutations in conserved motif A drastically alter RdRP fidelity, which can be either increased or decreased depending on the viral polymerase background. In the work presented here, we extend these studies to motif D, a region that forms the outer edge of the NTP entry channel where it may act as a nucleotide sensor to trigger active site closure. Crystallography, stopped-flow kinetics, quench-flow reactions, and infectious virus studies were used to characterize 15 engineered mutations in coxsackievirus B3 polymerase. Mutations that interfere with the transport of the metal A Mg(2+) ion into the active site had only minor effects on RdRP function, but the stacking interaction between Phe(364) and Pro(357), which is absolutely conserved in enteroviral polymerases, was found to be critical for processive elongation and virus growth. Mutating Phe(364) to tryptophan resulted in a genetically stable high fidelity virus variant with significantly reduced pathogenesis in mice. The data further illustrate the importance of the palm domain movement for RdRP active site closure and demonstrate that protein engineering can be used to alter viral polymerase function and attenuate virus growth and pathogenesis.

Structure-function relationships underlying the replication fidelity of viral RNA-dependent RNA polymerases.

UNLABELLED: Viral RNA-dependent RNA polymerases are considered to be low-fidelity enzymes, providing high mutation rates that allow for the rapid adaptation of RNA viruses to different host cell environments. Fidelity is tuned to provide the proper balance of virus replication rates, pathogenesis, and tissue tropism needed for virus growth. Using our structures of picornaviral polymerase-RNA elongation complexes, we have previously engineered more than a dozen coxsackievirus B3 polymerase mutations that significantly altered virus replication rates and in vivo fidelity and also provided a set of secondary adaptation mutations after tissue culture passage. Here we report a biochemical analysis of these mutations based on rapid stopped-flow kinetics to determine elongation rates and nucleotide discrimination factors. The data show a spatial separation of fidelity and replication rate effects within the polymerase structure. Mutations in the palm domain have the greatest effects on in vitro nucleotide discrimination, and these effects are strongly correlated with elongation rates and in vivo mutation frequencies, with faster polymerases having lower fidelity. Mutations located at the top of the finger domain, on the other hand, primarily affect elongation rates and have relatively minor effects on fidelity. Similar modulation effects are seen in poliovirus polymerase, an inherently lower-fidelity enzyme where analogous mutations increase nucleotide discrimination. These findings further our understanding of viral RNA-dependent RNA polymerase structure-function relationships and suggest that positive-strand RNA viruses retain a unique palm domain-based active-site closure mechanism to fine-tune replication fidelity. IMPORTANCE: Positive-strand RNA viruses represent a major class of human and animal pathogens with significant health and economic impacts. These viruses replicate by using a virally encoded RNA-dependent RNA polymerase enzyme that has low fidelity, generating many mutations that allow the rapid adaptation of these viruses to different tissue types and host cells. In this work, we use a structure-based approach to engineer mutations in viral polymerases and study their effects on in vitro nucleotide discrimination as well as virus growth and genome replication fidelity. These results show that mutation rates can be drastically increased or decreased as a result of single mutations at several key residues in the polymerase palm domain, and this can significantly attenuate virus growth in vivo. These findings provide a pathway for developing live attenuated virus vaccines based on engineering the polymerase to reduce virus fitness.

Crystal structures of the S. cerevisiae Spt6 core and C-terminal tandem SH2 domain.

The conserved and essential eukaryotic protein Spt6 functions in transcription elongation, chromatin maintenance, and RNA processing. Spt6 has three characterized functions. It is a histone chaperone capable of reassembling nucleosomes, a central component of transcription elongation complexes, and is required for recruitment of RNA processing factors to elongating RNA polymerase II (RNAPII). Here, we report multiple crystal structures of the 168-kDa Spt6 protein from Saccharomyces cerevisiae that together represent essentially all of the ordered sequence. Our two structures of the ∼900-residue core region reveal a series of putative nucleic acid and protein-protein interaction domains that fold into an elongated form that resembles the bacterial protein Tex. The similarity to a bacterial transcription factor suggests that the core domain performs nucleosome-independent activities, and as with Tex, we find that Spt6 binds DNA. Unlike Tex, however, the Spt6 S1 domain does not contribute to this activity. Crystal structures of the Spt6 C-terminal region reveal a tandem SH2 domain structure composed of two closely associated SH2 folds. One of these SH2 folds is cryptic, while the other shares striking structural similarity with metazoan SH2 domains and possesses structural features associated with the ability to bind phosphorylated substrates including phosphotyrosine. Binding studies with phosphopeptides that mimic the RNAPII C-terminal domain revealed affinities typical of other RNAPII C-terminal domain-binding proteins but did not indicate a specific interaction. Overall, these findings provide a structural foundation for understanding how Spt6 encodes several distinct functions within a single polypeptide chain.

Structure and biological importance of the Spn1-Spt6 interaction, and its regulatory role in nucleosome binding.

Eukaryotic transcription and mRNA processing depend upon the coordinated interactions of many proteins, including Spn1 and Spt6, which are conserved across eukaryotes, are essential for viability, and associate with each other in some of their biologically important contexts. Here we report crystal structures of the Spn1 core alone and in complex with the binding determinant of Spt6. Mutating interface residues greatly diminishes binding in vitro and causes strong phenotypes in vivo, including a defect in maintaining repressive chromatin. Overexpression of Spn1 partially suppresses the defects caused by an spt6 mutation affecting the Spn1 interface, indicating that the Spn1-Spt6 interaction is important for managing chromatin. Spt6 binds nucleosomes directly in vitro, and this interaction is blocked by Spn1, providing further mechanistic insight into the function of the interaction. These data thereby reveal the structural and biochemical bases of molecular interactions that function in the maintenance of chromatin structure.