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We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-l-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-l-tyrosine. The 1.7 Å crystal structure...
Splicing requires reversible phosphorylation of serine/arginine-rich (SR) proteins, which direct splice site selection in eukaryotic mRNA. These phosphorylation events are dependent on SR protein (SRPK) and cdc2-like kinase (CLK) families. SRPK1 phosphorylation of splicing factors is restricted by a specific docking interaction whereas CLK activity is less constrained. To understand functional differences...
Peptides comprising residues 106–126 of the human prion protein (PrP) exhibit many features of the full-length protein. PrP(106–126) induces apoptosis in neurons, forms fibrillar aggregates, and can mediate the conversion of native cellular PrP (PrP C ) to the scrapie form (PrP Sc ). Despite a wide range of biochemical and biophysical studies on this peptide, including investigation...
The neural cell adhesion molecule L1 participates in homophilic interactions important for axon guidance and neuronal development. The structural details of homophilic adhesion mediated by L1 and other immunoglobulin superfamily members containing an N-terminal horseshoe arrangement of four immunoglobulin-like domains are unknown. Here we used cryo-electron tomography to study liposomes to which intact...
The general transcription factor TFIID is a large multisubunit complex required for the transcription of most protein-encoding genes by RNA polymerase II. Taking advantage of a TFIID preparation partially depleted in the initiator-binding Taf2p subunit, we determined the conformational and biochemical variations of the complex by electron tomography and cryo-electron microscopy of single molecules...
Messing et al. (2009) report the homodimeric structure of the Bdellovibrio bacteriovorus RppH pyrophosphohydrolase, which hydrolyzes the mRNA 5′ triphosphate to initiate bacterial mRNA decay. These structures reveal insights into BdRppH substrate recognition and analogies to eukaryotic decapping enzymes.
The human Hrs and STAM proteins comprise the ESCRT-0 complex, which sorts ubiquitinated cell surface receptors to lysosomes for degradation. Here we report a model for the complete ESCRT-0 complex based on the crystal structure of the Hrs-STAM core complex, previously solved domain structures, hydrodynamic measurements, and Monte Carlo simulations. ESCRT-0 expressed in insect cells has a hydrodynamic...
γ-Secretase is an intramembrane protease complex that mediates the Notch signaling pathway and the production of amyloid β-proteins. As such, this enzyme has emerged as an important target for development of novel therapeutics for Alzheimer disease and cancer. Great progress has been made in the identification and characterization of the membrane complex and its biological functions. One major challenge...
Use of heavy atoms to calculate the phases in protein X-ray crystallography has now been extended to include site-specific in vivo incorporation of 3-iodo-tyrosine, enabled by the use of an archaeal aminoacyl-tRNA synthetase (Sakamoto et al., 2009).
Until recently, the mechanism of mRNA decay in bacteria was thought to be different from that of eukaryotes. This paradigm changed with the discovery that RppH (ORF176/NudH/YgdP), an Escherichia coli enzyme that belongs to the Nudix superfamily, is an RNA pyrophosphohydrolase that initiates mRNA decay by cleaving pyrophosphate from the 5′-triphosphate. Here we report the 1.9 Å resolution structure...
Mauldin et al. (2009) use NMR to show that drug binding can break up collective protein motions necessary for function. We discuss their findings in the context of drug discovery in pharmaceutical research.
Among methyltransferases, KsgA and the reaction it catalyzes are conserved throughout evolution. However, the specifics of substrate recognition by the enzyme remain unknown. Here we report structures of Aquifex aeolicus KsgA, in its ligand-free form, in complex with RNA, and in complex with both RNA and S-adenosylhomocysteine (SAH, reaction product of cofactor S-adenosylmethionine), revealing critical...
Integral membrane proteins remain a significant challenge to structural studies by solution NMR spectroscopy. This is due not only to spectral complexity, but also because the effects of slow molecular reorientation are exacerbated by the need to solubilize the protein in aqueous detergent micelles. These assemblies can be quite large and require deuteration for optimal use of the TROSY effect. In...
Nuclear pore complexes (NPCs) are selectively gated pathways between nucleoplasm and cytoplasm. Whereas small molecules can diffuse freely through NPCs, large molecules (>40 kD) can pass only when bound to transport receptors. The NPC central channel is filled with disordered proteins, rich in phenylalanine-glycine (FG) repeats, referred to as FG-nups. Our simulations, carried out at coarse-grained...
Intramembrane proteases, which cleave transmembrane (TM) helices, participate in numerous biological processes encompassing all branches of life. Several crystallographic structures of Escherichia coli GlpG rhomboid protease have been determined. In order to understand GlpG dynamics and lipid interactions in a native-like environment, we have examined the molecular dynamics of wild-type and mutant...
Vps4 is a AAA ATPase that mediates endosomal membrane protein sorting. It is also a host factor hijacked by a diverse set of clinically important viruses, including HIV and Ebola, to facilitate viral budding. Here we present the three-dimensional structure of the hydrolysis-defective Vps4p E233Q mutant. Single-particle analysis, multiangle laser light scattering, and the docking of independently...
In plants, unorthodox multisubunit RNA polymerases (RNAPs) play key roles in small interfering RNA (siRNA) genesis and function. In a recent issue of Molecular Cell, Ream et al. (2009) established a 12-subunit composition for Arabidopsis RNAPIV and RNAPV. Subunit and sequence divergence between RNAPIV-V and RNAPI-III suggests significant functional deviation of these intriguing RNAPs.
We review insights from computational studies of affinities of ligands binding to proteins. The power of structural biology is in translating knowledge of protein structures into insights about their forces, binding, and mechanisms. However, the complementary power of computer modeling is in showing “the rest of the story” (i.e., how motions and ensembles and alternative conformers and the entropies...
The NMR structures of Corynebacterium glutamicum OdhI described by Barthe et al. in this issue of Structure reveal a major conformational rearrangement upon phosphorylation, suggesting an autoinhibition mechanism that accounts for the functional properties of the protein as a regulatory switch in glutamate metabolism.
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