Zhu Liu

Structural basis of N6-adenosine methylation by the METTL3-METTL14 complex

Chemical modifications of RNA have essential roles in a vast range of cellular processes. N6-methyladenosine (m6A) is an abundant internal modification in messenger RNA and long non-coding RNA that can be dynamically added and removed by RNA methyltransferases (MTases) and demethylases, respectively. An MTase complex comprising methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) efficiently catalyses methyl group transfer. In contrast to the well-studied DNA MTase, the exact roles of these two RNA MTases in the complex remain to be elucidated. Here we report the crystal structures of the METTL3–METTL14 heterodimer with MTase domains in the ligand-free, S-adenosyl methionine (AdoMet)-bound and S-adenosyl homocysteine (AdoHcy)-bound states, with resolutions of 1.9, 1.71 and 1.61 Å, respectively. Both METTL3 and METTL14 adopt a class I MTase fold and they interact with each other via an extensive hydrogen bonding network, generating a positively charged groove. Notably, AdoMet was observed in only the METTL3 pocket and not in METTL14. Combined with biochemical analysis, these results suggest that in the m6A MTase complex, METTL3 primarily functions as the catalytic core, while METTL14 serves as an RNA-binding platform, reminiscent of the target recognition domain of DNA N6-adenine MTase. This structural information provides an important framework for the functional investigation of m6A.

Noncovalent Dimerization of Ubiquitin

Another kind of dynamics: Ubiquitin noncovalently dimerizes with a dissociation constant of approximately 5 mM. The two subunits adopt an array of relative orientations, utilizing an interface also for binding to other proteins (see picture). Quaternary fluctuation among members of the dimer ensemble constitutes a different kind of dynamics that complements the tertiary dynamics of each ubiquitin subunit.

Transient protein-protein interactions visualized by solution NMR

Proteins interact with each other to establish their identities in cell. The affinities for the interactions span more than ten orders of magnitude, and KD values in μM-mM regimen are considered transient and are important in cell signaling. Solution NMR including diamagnetic and paramagnetic techniques has enabled atomic-resolution depictions of transient protein-protein interactions. Diamagnetic NMR allows characterization of protein complexes with KD values up to several mM, whereas ultraweak and fleeting complexes can be modeled with the use of paramagnetic NMR especially paramagnetic relaxation enhancement (PRE). When tackling ever-larger protein complexes, PRE can be particularly useful in providing long-range intermolecular distance restraints. As NMR measurements are averaged over the ensemble of complex structures, structural information for dynamic protein-protein interactions besides the stereospecific one can often be extracted. Herein the protein interaction dynamics are exemplified by encounter complexes, alternative binding modes, and coupled binding/folding of intrinsically disordered proteins. Further integration of NMR with other biophysical techniques should allow better visualization of transient protein-protein interactions. In particular, single-molecule data may facilitate the interpretation of ensemble-averaged NMR data. Though same structures of proteins and protein complexes were found in cell as in diluted solution, we anticipate that the dynamics of transient protein protein-protein interactions be different, which awaits awaits exploration by NMR. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions.

Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition

A polyubiquitin comprises multiple covalently linked ubiquitins and recognizes myriad targets. Free or bound to ligands, polyubiquitins are found in different arrangements of ubiquitin subunits. To understand the structural basis for polyubiquitin quaternary plasticity and to explore the target recognition mechanism, we characterize the conformational space of Lys63-linked diubiquitin (K63-Ub2). Refining against inter-subunit paramagnetic NMR data, we show that free K63-Ub2 exists as a dynamic ensemble comprising multiple closed and open quaternary states. The quaternary dynamics enables K63-Ub2 to be specifically recognized in a variety of signaling pathways. When binding to a target protein, one of the preexisting quaternary states is selected and stabilized. A point mutation that shifts the equilibrium between the different states modulates the binding affinities towards K63-Ub2 ligands. This conformational selection mechanism at the quaternary level may be used by polyubiquitins of different lengths and linkages for target recognition. DOI: http://dx.doi.org/10.7554/eLife.05767.001

Corrigendum: Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex

This corrects the article DOI: 10.1038/nature18298

Modeling protein excited-state structures from "over-length" chemical cross-links

Chemical cross-linking coupled with mass spectroscopy (CXMS) provides proximity information for the cross-linked residues and is used increasingly for modeling protein structures. However, experimentally identified cross-links are sometimes incompatible with the known structure of a protein, as the distance calculated between the cross-linked residues far exceeds the maximum length of the cross-linker. The discrepancies may persist even after eliminating potentially false cross-links and excluding intermolecular ones. Thus the “over-length” cross-links may arise from alternative excited-state conformation of the protein. Here we present a method and associated software DynaXL for visualizing the ensemble structures of multidomain proteins based on intramolecular cross-links identified by mass spectrometry with high confidence. Representing the cross-linkers and cross-linking reactions explicitly, we show that the protein excited-state structure can be modeled with as few as two over-length cross-links. We demonstrate the generality of our method with three systems: calmodulin, enzyme I, and glutamine-binding protein, and we show that these proteins alternate between different conformations for interacting with other proteins and ligands. Taken together, the over-length chemical cross-links contain valuable information about protein dynamics, and our findings here illustrate the relationship between dynamic domain movement and protein function.

Subtle Dynamics of holo Glutamine Binding Protein Revealed with a Rigid Paramagnetic Probe

Bacterial periplasmic binding proteins (PBPs) are involved in the translocation of small molecules in the periplasm. To unload, the two domains of a PBP open up, allowing the ligand to exit. However, it is not clear whether there are dynamics near the binding site which can facilitate the rapid dissociation of a ligand. To visualize such dynamics, we utilized paramagnetic relaxation enhancement (PRE) NMR and introduced a rigid paramagnetic probe to a PBP, glutamine-binding protein (QBP) with its cognate ligand bound. A paramagnetic Cu(II) ion is sandwiched between an engineered di-histidine motif at a helix and an NTA capping molecule. The afforded paramagnetic probe is so rigid that PRE values calculated from a single structure of holo QBP largely agree with the observed values. The remaining PRE discrepancies, however, manifest dynamics of a loop in the opposite domain from the paramagnetic probe. This loop packs against the glutamine ligand in the holo QBP and undergoes fluctuations upon ligand dissociation, as assessed by steered molecular dynamics simulations. As such, the loop dynamics, occurring for a small population in nanosecond to microsecond time scale, may be related to the ligand dissociation process. The rigid paramagnetic probe described herein can be grafted to other protein systems for structure and dynamics studies.

Ubiquitin S65 phosphorylation engenders a pH-sensitive conformational switch

Significance Ubiquitination and phosphorylation are the two most important protein posttranslational modifications and cell signals. Ubiquitin can be specifically phosphorylated at S65, and the finding here suggests a general functional role for Ub phosphorylation. We show that subtle fluctuation near physiological pH can affect the protonation status of the S65 phosphoryl group and modulate the structure of the ubiquitin monomer and polyubiquitin. It is known that cellular pH varies among organelles and changes under physiological and pathological conditions. Because ubiquitin is involved in myriad aspects of cell biology, a pH-sensitive conformational switch acquired upon S65 phosphorylation would allow phosphorylated ubiquitin to interact with different target proteins upon environmental cues. It would also enable cross-talk between ubiquitination and phosphorylation signals. Ubiquitin (Ub) is an important signaling protein. Recent studies have shown that Ub can be enzymatically phosphorylated at S65, and that the resulting pUb exhibits two conformational states—a relaxed state and a retracted state. However, crystallization efforts have yielded only the structure for the relaxed state, which was found similar to that of unmodified Ub. Here we present the solution structures of pUb in both states obtained through refinement against state-specific NMR restraints. We show that the retracted state differs from the relaxed state by the retraction of the last β-strand and by the extension of the second α-helix. Further, we show that at 7.2, the pKa value for the phosphoryl group in the relaxed state is higher by 1.4 units than that in the retracted state. Consequently, pUb exists in equilibrium between protonated and deprotonated forms and between retracted and relaxed states, with protonated/relaxed species enriched at slightly acidic pH and deprotonated/retracted species enriched at slightly basic pH. The heterogeneity of pUb explains the inability of phosphomimetic mutants to fully mimic pUb. The pH-sensitive conformational switch is likely preserved for polyubiquitin, as single-molecule FRET data indicate that pH change leads to quaternary rearrangement of a phosphorylated K63-linked diubiquitin. Because cellular pH varies among compartments and changes upon pathophysiological insults, our finding suggests that pH and Ub phosphorylation confer additional target specificities and enable an additional layer of modulation for Ub signals.

Characterizing Protein Dynamics with Integrative Use of Bulk and Single-Molecule Techniques

A protein dynamically samples multiple conformations, and the conformational dynamics enables protein function. Most biophysical measurements are ensemble-based, with the observables averaged over all members of the ensemble. Though attainable, the decomposition of the observables to the constituent conformational states can be computationally expensive and ambiguous. Here we show that the incorporation of single-molecule fluorescence resonance energy transfer (smFRET) data resolves the ambiguity and affords protein ensemble structures that are more precise and accurate. Using K63-linked diubiquitin, we characterize the dynamic domain arrangements of the model system, with the use of chemical cross-linking coupled with mass spectrometry (CXMS), small-angle X-ray scattering (SAXS), and smFRET techniques. CXMS allows the modeling of protein conformational states that are alternatives to the crystal structure. SAXS provides ensemble-averaged low-resolution shape information. Importantly, smFRET affords state-specific populations, and the FRET distances validate the ensemble structures obtained by refining against CXMS and SAXS restraints. Together, the integrative use of bulk and single-molecule techniques affords better insight into protein dynamics and shall be widely implemented in structural biology.

Ensemble structure description of Lys63-linked diubiquitin

The data described herein are related to the article entitled “Lys63-linked ubiquitin chain adopts multiple conformational states for specific target recognition” [1], and to the coordinates for the ensemble structure of Lys63-linked diubiquitin (PDB code 2N2K). A Lys63-linked diubiquitin exists in three conformational states with different orientations for the two subunits, each responsible for binding to a target protein and encoding a specific cell signal. An atomic entry in the ensemble structure file consists multiple lines, representing alternative locations of the atom and recapitulating the dynamics of the protein. Experimental details about obtaining strictly intramolecular paramagnetic restraints and determining the relative occupancies of the conformational states are presented. The experimental design and procedures in this Data article can be useful for characterizing the structure and dynamics of other multi-domain proteins.