Crystal structure of TET2-DNA complex: insight into TET-mediated 5mC oxidation.

TET proteins oxidize 5-methylcytosine (5mC) on DNA and play important roles in various biological processes. Mutations of TET2 are frequently observed in myeloid malignance. Here, we present the crystal structure of human TET2 bound to methylated DNA at 2.02 Å resolution. The structure shows that two zinc fingers bring the Cys-rich and DSBH domains together to form a compact catalytic domain. The Cys-rich domain stabilizes the DNA above the DSBH core. TET2 specifically recognizes CpG dinucleotide and shows substrate preference for 5mC in a CpG context. 5mC is inserted into the catalytic cavity with the methyl group orientated to catalytic Fe(II) for reaction. The methyl group is not involved in TET2-DNA contacts so that the catalytic cavity allows TET2 to accommodate 5mC derivatives for further oxidation. Mutations of Fe(II)/NOG-chelating, DNA-interacting, and zinc-chelating residues are frequently observed in human cancers. Our studies provide a structural basis for understanding the mechanisms of TET-mediated 5mC oxidation.

Multi-curve fitting and tubulin-lattice signal removal for structure determination of large microtubule-based motors.

Revealing high-resolution structures of microtubule-associated proteins (MAPs) is critical for understanding their fundamental roles in various cellular activities, such as cell motility and intracellular cargo transport. Nevertheless, large flexible molecular motors that dynamically bind and release microtubule networks are challenging for cryo-electron microscopy (cryo-EM). Traditional structure determination of MAPs bound to microtubules needs alignment information from the reconstruction of microtubules, which cannot be readily applied to large MAPs without a fixed binding pattern. Here, we developed a comprehensive approach to estimate the microtubule networks (multi-curve fitting), model the tubulin-lattice signals, and remove them (tubulin-lattice subtraction) from the raw cryo-EM micrographs. The approach does not require an ordered binding pattern of MAPs on microtubules, nor does it need a reconstruction of the microtubules. We demonstrated the capability of our approach using the reconstituted outer-arm dynein (OAD) bound to microtubule doublets. The tubulin-lattice subtraction improves the OAD alignment, thus leading to high-resolution reconstructions. In addition, the multi-curve fitting approach provides an accurate automatic alternative method to pick or segment filaments in 2D images and potentially in 3D tomograms. The accuracy of our approach has been demonstrated by using several other biological filaments. Our work provides a new tool to determine high-resolution structures of large MAPs bound to curved microtubule networks.

Structural insight into substrate preference for TET-mediated oxidation.

DNA methylation is an important epigenetic modification. Ten-eleven translocation (TET) proteins are involved in DNA demethylation through iteratively oxidizing 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Here we show that human TET1 and TET2 are more active on 5mC-DNA than 5hmC/5fC-DNA substrates. We determine the crystal structures of TET2-5hmC-DNA and TET2-5fC-DNA complexes at 1.80 Å and 1.97 Å resolution, respectively. The cytosine portion of 5hmC/5fC is specifically recognized by TET2 in a manner similar to that of 5mC in the TET2-5mC-DNA structure, and the pyrimidine base of 5mC/5hmC/5fC adopts an almost identical conformation within the catalytic cavity. However, the hydroxyl group of 5hmC and carbonyl group of 5fC face towards the opposite direction because the hydroxymethyl group of 5hmC and formyl group of 5fC adopt restrained conformations through forming hydrogen bonds with the 1-carboxylate of NOG and N4 exocyclic nitrogen of cytosine, respectively. Biochemical analyses indicate that the substrate preference of TET2 results from the different efficiencies of hydrogen abstraction in TET2-mediated oxidation. The restrained conformation of 5hmC and 5fC within the catalytic cavity may prevent their abstractable hydrogen(s) adopting a favourable orientation for hydrogen abstraction and thus result in low catalytic efficiency. Our studies demonstrate that the substrate preference of TET2 results from the intrinsic value of its substrates at their 5mC derivative groups and suggest that 5hmC is relatively stable and less prone to further oxidation by TET proteins. Therefore, TET proteins are evolutionarily tuned to be less reactive towards 5hmC and facilitate the generation of 5hmC as a potentially stable mark for regulatory functions.

High-Resolution Structural Analysis of Dyneins by Cryo-electron Microscopy.

Cryo-electron microscopy (cryo-EM) has become the mainstream technique for studying macromolecular structures. Determining the structures of protein complexes is more accessible to structural biologists than ever before. Nevertheless, obtaining high-resolution structures of molecular motors like dynein is still an extremely challenging goal due to their troublesome behaviors in ice, their exceedingly flexible conformations, and their intricate architectures. Dynein is a large molecular machine that drives the movement of many essential cellular cargos and is also the key force generator that powers ciliary motility. High-resolution structural information of dyneins in different states is critical for the in-depth mechanistic understanding of their roles in cells. Here, we summarize the cryo-EM approaches that we have used to study the structures of outer-arm dynein arrays bound to microtubule doublets. Our approaches can be applied to other similar structures and further optimized to deal with even more complicated targets.

Cryo-EM structure of an active central apparatus.

Accurately regulated ciliary beating in time and space is critical for diverse cellular activities, which impact the survival and development of nearly all eukaryotic species. An essential beating regulator is the conserved central apparatus (CA) of motile cilia, composed of a pair of microtubules (C1 and C2) associated with hundreds of protein subunits per repeating unit. It is largely unclear how the CA plays its regulatory roles in ciliary motility. Here, we present high-resolution structures of Chlamydomonas reinhardtii CA by cryo-electron microscopy (cryo-EM) and its dynamic conformational behavior at multiple scales. The structures show how functionally related projection proteins of CA are clustered onto a spring-shaped scaffold of armadillo-repeat proteins, facilitated by elongated rachis-like proteins. The two halves of the CA are brought together by elastic chain-like bridge proteins to achieve coordinated activities. We captured an array of kinesin-like protein (KLP1) in two different stepping states, which are actively correlated with beating wave propagation of cilia. These findings establish a structural framework for understanding the role of the CA in cilia.

Structures of outer-arm dynein array on microtubule doublet reveal a motor coordination mechanism.

Thousands of outer-arm dyneins (OADs) are arrayed in the axoneme to drive a rhythmic ciliary beat. Coordination among multiple OADs is essential for generating mechanical forces to bend microtubule doublets (MTDs). Using electron microscopy, we determined high-resolution structures of Tetrahymena thermophila OAD arrays bound to MTDs in two different states. OAD preferentially binds to MTD protofilaments with a pattern resembling the native tracks for its distinct microtubule-binding domains. Upon MTD binding, free OADs are induced to adopt a stable parallel conformation, primed for array formation. Extensive tail-to-head (TTH) interactions between OADs are observed, which need to be broken for ATP turnover by the dynein motor. We propose that OADs in an array sequentially hydrolyze ATP to slide the MTDs. ATP hydrolysis in turn relaxes the TTH interfaces to effect free nucleotide cycles of downstream OADs. These findings lead to a model explaining how conformational changes in the axoneme produce coordinated action of dyneins.

Cryo-EM structure of human ATR-ATRIP complex.

ATR (ataxia telangiectasia-mutated and Rad3-related) protein kinase and ATRIP (ATR-interacting protein) form a complex and play a critical role in response to replication stress and DNA damage. Here, we determined the cryo-electron microscopy (EM) structure of the human ATR-ATRIP complex at 4.7 Å resolution and built an atomic model of the C-terminal catalytic core of ATR (residues 1 521-2 644) at 3.9 Å resolution. The complex adopts a hollow "heart" shape, consisting of two ATR monomers in distinct conformations. The EM map for ATRIP reveals 14 HEAT repeats in an extended "S" shape. The conformational flexibility of ATR allows ATRIP to properly lock the N-termini of the two ATR monomers to favor ATR-ATRIP complex formation and functional diversity. The isolated "head-head" and "tail-tail" each adopts a pseudo 2-fold symmetry. The catalytic pockets face outward and substrate access is not restricted by inhibitory elements. Our studies provide a structural basis for understanding the assembly of the ATR-ATRIP complex and a framework for characterizing ATR-mediated DNA repair pathways.