Chemical modifications of RNA have essential roles in a vast range of cellular processes. N(6)-methyladenosine (m(6)A) 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 m(6)A 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 N(6)-adenine MTase. This structural information provides an important framework for the functional investigation of m(6)A.

Kinetic data on a number of protein-protein associations have provided evidence for the initial formation of a pre-equilibrium encounter complex that subsequently relaxes to the final stereospecific complex. Site-directed mutagenesis and brownian dynamics simulations have suggested that the rate of association can be modulated by perturbations in charge distribution outside the direct interaction surfaces. Furthermore, rate enhancement through non-specific binding may occur by either a reduction in dimensionality or the presence of a short-range, non-specific attractive potential. Here, using paramagnetic relaxation enhancement, we directly demonstrate the existence and visualize the distribution of an ensemble of transient, non-specific encounter complexes under equilibrium conditions for a relatively weak protein-protein complex between the amino-terminal domain of enzyme I and the phosphocarrier protein HPr. Neither the stereospecific complex alone nor any single alternative conformation can account fully for the intermolecular paramagnetic relaxation enhancement data. Restrained rigid-body simulated annealing refinement against the paramagnetic relaxation enhancement data enables us to obtain an atomic probability distribution map of the non-specific encounter complex ensemble that qualitatively correlates with the electrostatic surface potentials on the interacting proteins. Qualitatively similar results are presented for two other protein-protein complexes.

The myristoylated matrix protein (myr-MA) of HIV functions as a regulator of intracellular localization, targeting the Gag precursor polyprotein to lipid rafts in the plasma membrane during virus assembly and dissociating from the membrane during infectivity for nuclear targeting of the preintegration complex. Membrane release is triggered by proteolytic cleavage of Gag, and it has, until now, been believed that proteolysis induces a conformational change in myr-MA that sequesters the myristyl group. NMR studies reported here reveal that myr-MA adopts myr-exposed [myr(e)] and -sequestered [myr(s)] states, as anticipated. Unexpectedly, the tertiary structures of the protein in both states are very similar, with the sequestered myristyl group occupying a cavity that requires only minor conformational adjustments for insertion. In addition, myristate exposure is coupled with trimerization, with the myristyl group sequestered in the monomer and exposed in the trimer (Kassoc ‫؍‬ 2.5 ؎ 0.6 ؋ 10 8 M ؊2 ). The equilibrium constant is shifted Ϸ20-fold toward the trimeric, myristate-exposed species in a Gag-like construct that includes the capsid domain, indicating that exposure is enhanced by Gag subdomains that promote self-association. Our findings indicate that the HIV-1 myristyl switch is regulated not by mechanically induced conformational changes, as observed for other myristyl switches, but instead by entropic modulation of a preexisting equilibrium.

Large-scale domain rearrangements in proteins have long been recognized to have a critical function in ligand binding and recognition, catalysis and regulation. Crystal structures have provided a static picture of the apo (usually open) and holo usually closed) states. The general question arises as to whether the apo state exists as a single species in which the closed state is energetically inaccessible and interdomain rearrangement is induced by ligand or substrate binding, or whether the predominantly open form already coexists in rapid equilibrium with a minor closed species. The maltose-binding protein (MBP), a member of the bacterial periplasmic binding protein family, provides a model system for investigating this problem because it has been the subject of extensive studies by crystallography, NMR and other biophysical techniques. Here we show that although paramagnetic relaxation enhancement (PRE) data for the sugar-bound form are consistent with the crystal structure of holo MBP, the PRE data for the apo state are indicative of a rapidly exchanging mixture (ns to mus regime) of a predominantly ( approximately 95%) open form (represented by the apo crystal structure) and a minor (approximately 5%) partially closed species. Using ensemble simulated annealing refinement against the PRE data we are able to determine a ensemble average structure of the minor apo species and show that it is distinct from the sugar-bound state.

The use of 1 H transverse paramagnetic relaxation enhancement (PRE) has seen a resurgence in recent years as method for providing long-range distance information for structural studies and as a probe of large amplitude motions and lowly populated transient intermediates in macromolecular association. In this paper we discuss various practical aspects pertaining to accurate measurement of PRE 1 H transverse relaxation rates (Γ 2 ). We first show that accurate Γ 2 rates can be obtained from a two time-point measurement without requiring any fitting procedures or complicated error estimations, and no additional accuracy is achieved from multiple time-point measurements recorded in the same experiment time. Optimal setting of the two time-points that minimize experimental errors is also discussed. Next we show that the simplistic single time-point measurement that has been commonly used in the literature, can substantially underestimate the true value of Γ 2 , unless a relatively long repetition delay is employed. We then examine the field dependence of Γ 2 , and show that Γ 2 exhibits only a very weak field dependence at high magnetic fields typically employed in macromolecular studies. The theoretical basis for this observation is discussed. Finally, we investigate the impact of contamination of the paramagnetic sample by trace amounts (≤5%) of the corresponding diamagnetic species on the accuracy of Γ 2 measurements. Errors in Γ 2 introduced by such diamagnetic contamination are potentially sizeable, but can be significantly reduced by using a relatively short time interval for the two time-point Γ 2 measurement.

Recent advances in the use of paramagnetic relaxation enhancement (PRE) in structure refinement and in the analysis of transient dynamic processes involved in macromolecular complex formation are presented. In the slow exchange regime, we show, using the SRY/DNA complex as an example, that the PRE provides a powerful tool that can lead to significant increases in the reliability and accuracy of NMR structure determinations. Refinement necessitates the use of an ensemble representation of the paramagnetic center and a model free extension of the Solomon-Bloembergen equations. In the fast exchange regime, the PRE provides insight into dynamic processes and the existence of transient, low population intermediate species. The PRE allows one to characterize dynamic non-specific binding of a protein to DNA; to directly demonstrate that the search process whereby a transcription factor locates its specific DNA target site involves both intramolecular (sliding) and intermolecular (hopping and intersegment transfer) translocation; and to detect and visualize the distribution of an ensemble of transient encounter complexes in protein-protein association.

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The capsid protein (CA) of the mature human immunodeficiency virus (HIV) contains an N-terminal beta-hairpin that is essential for formation of the capsid core particle. CA is generated by proteolytic cleavage of the Gag precursor polyprotein during viral maturation. We have determined the NMR structure of a 283-residue N-terminal fragment of immature HIV-1 Gag (Gag(283)), which includes the intact matrix (MA) and N-terminal capsid (CA(N)) domains. The beta-hairpin is unfolded in Gag(283), consistent with the proposal that hairpin formation occurs subsequent to proteolytic cleavage of Gag, triggering capsid assembly. Comparison of the immature and mature CA(N) structures reveals that beta-hairpin formation induces a approximately 2 A displacement of helix 6 and a concomitant displacement of the cyclophylin-A (CypA)-binding loop, suggesting a possible allosteric mechanism for CypA-mediated destabilization of the capsid particle during infectivity.