Journal of structural and functional genomics最新文献

Rotaviruses are a global health concern causing severe childhood diarrhea. Though the mechanisms mediating rotavirus cell entry require further characterisation, it is acknowledged that an essential and critical step in rotavirus infection is the virus cell attachment via initial recognition of cell surface carbohydrate-containing receptors. This review summarises the current knowledge about recognition of glycan receptors by rotaviruses.

The N (1)-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m(1)A58 methyltransferase TrmI, using S-adenosyl-L-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.

Cryo-transmission electron microscopy (Cryo-TEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three-dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.

Phosphorylation and acetylation are the most prevalent post-translational modifications (PTMs) detected in not only eukaryotes but also bacteria. We performed phosphoproteome and acetylome analyses of proteins from an extremely thermophilic eubacterium Thermus thermophilus HB8, and identified numerous phosphorylation and acetylation sites. To facilitate the elucidation of the structural aspects of these PTM events, we mapped the PTM sites on the known tertiary structures for the respective proteins and their homologs. Wu et al. (Mol Cell Proteomics 12:2701-2713, 2013) recently reported phosphoproteome analysis of proteins from T. thermophilus HB27. Therefore, we assessed the structural characteristics of these phosphorylation and acetylation sites on the tertiary structures of the identified proteins or their homologs. Our study revealed that many of the identified phosphosites are in close proximity to bound ligands, i.e., the numbers of 'nearby' and 'peripheral' phosphorylation sites represent 56 % (48/86 sites) of total identified phosphorylation sites. In addition, approximately 60 % of all phosphosites exhibited <10 % accessible surface area of their side chains, suggesting some structural rearrangement is required for phosphoryl transfer by kinases. Our findings also indicate that phosphorylation of a residue occurs more frequently at a flexible region of the protein, whereas lysine acetylation occurs more frequently in an ordered structure.

The Sec translocon facilitates transportation of newly synthesized polypeptides from the cytoplasm to the lumen/periplasm across the phospholipid membrane. Although the polypeptide-conducting machinery is formed by the SecYEG-SecA complex in bacteria, its transportation efficiency is markedly enhanced by SecDF. A previous study suggested that SecDF assumes at least two conformations differing by a 120° rotation in the spatial orientation of the P1 head subdomain to the rigid base, and that the conformational dynamics plays a critical role in polypeptide translocation. Here we addressed this hypothesis by analyzing the 3D structure of SecDF using electron tomography and single particle reconstruction. Reconstruction of wt SecDF showed two major conformations; one resembles the crystal structure of full-length SecDF (F-form structure), while the other is similar to the hypothetical structural variant based on the crystal structure of the isolated P1 domain (I-form structure). The transmembrane domain of the I-form structure has a scissor like cleft open to the periplasmic side. We also report the structure of a double cysteine mutant designed to constrain SecDF to the I-form. This reconstruction has a protrusion at the periplasmic end that nicely fits the orientation of P1 in the I-from. These results provide firm evidence for the occurrence of the I-form in solution and support the proposed F- to I-transition of wt SecDF during polypeptide translocation.

A reliable and easy to use manual dispensing system has been developed for the in meso membrane protein crystallization method. The system consists of a stepping motor-based dispenser with a new microsyringe system for dispensing, which allows us to deliver any desired volume of highly viscous lipidic mesophase in the range from ~50 to at least ~200 nl. The average, standard deviation, and coefficient of variation of 20 repeated deliveries of 50 nl cubic phase were comparable to those of a current robotic dispensing. Moreover, the bottom faces of boluses delivered to the glass crystallization plate were reproducibly circular in shape, and their centers were within about 100 μm from the center of the crystallization well. The system was useful for crystallizing membrane and soluble proteins in meso.

Protein synthesis requires accurate charging of tRNA with cognate amino acid as catalyzed by aminoacyl-tRNA synthetases. Crystal structures of tyrosyl-tRNA synthetase (YRSs) show remarkably diverse conformations for the KMSKS loop, hitherto classified as "open" and "closed". This traditional classification implied that the KMSKS loop adopts different conformations depending on occupancy of active site pocket. Our structural analyses of evolutionarily derived ensemble of differentially ligated YRSs using quantitative structural criterion demonstrate intrinsic conformational heterogeneity in KMSKS loop that is independent of occupancy of active site. Differential centroid distance analyses between KMSKS motif and Rossmann fold domain reveal an intriguing bimodal distribution. These insights have been used for a more consistent re-classification of YRS conformations as either compact or extended. Our data not only reflect inherent dynamics within the conformational landscape of KMSKS loops, but also have implications for structure-based drug design efforts.

Malaria parasites inevitably develop drug resistance to anti-malarials over time. Hence the immediacy for discovering new chemical scaffolds to include in combination malaria drug therapy. The desirable attributes of new chemotherapeutic agents currently include activity against both liver and blood stage malaria parasites. One such recently discovered compound called cladosporin abrogates parasite growth via inhibition of Plasmodium falciparum lysyl-tRNA synthetase (PfKRS), an enzyme central to protein translation. Here, we present crystal structure of ternary PfKRS-lysine-cladosporin (PfKRS-K-C) complex that reveals cladosporin's remarkable ability to mimic the natural substrate adenosine and thereby colonize PfKRS active site. The isocoumarin fragment of cladosporin sandwiches between critical adenine-recognizing residues while its pyran ring fits snugly in the ribose-recognizing cavity. PfKRS-K-C structure highlights ample space within PfKRS active site for further chemical derivatization of cladosporin. Such derivatives may be useful against additional human pathogens that retain high conservation in cladosporin chelating residues within their lysyl-tRNA synthetase.

We report a 2.0 Å structure of the CAE31940 protein, a proteobacterial NMT1/THI5-like domain-containing protein. We also discuss the primary and tertiary structure similarity with its homologs. The highly conserved FGGXMP motif was identified in CAE31940, which corresponds to the GCCCX motif located in the vicinity of the active center characteristic for THi5-like proteins found in yeast. This suggests that the FGGXMP motif may be a unique hallmark of proteobacterial NMT1/THI5-like proteins.

Sequence analysis of large protein families can produce sub-clusters even within the same family. In some cases, it is of interest to know precisely which amino acid position variations are most responsible for driving separation into sub-clusters. In large protein families composed of large proteins, it can be quite challenging to assign the relative importance to specific amino acid positions. Principal components analysis (PCA) is ideal for such a task, since the problem is posed in a large variable space, i.e. the number of amino acids that make up the protein sequence, and PCA is powerful at reducing the dimensionality of complex problems by projecting the data into an eigenspace that represents the directions of greatest variation. However, PCA of aligned protein sequence families is complicated by the fact that protein sequences are traditionally represented by single letter alphabetic codes, whereas PCA of protein sequence families requires conversion of sequence information into a numerical representation. Here, we introduce a new amino acid sequence conversion algorithm optimized for PCA data input. The method is demonstrated using a small artificial dataset to illustrate the characteristics and performance of the algorithm, as well as a small protein sequence family consisting of nine members, COG2263, and finally with a large protein sequence family, Pfam04237, which contains more than 1,800 sequences that group into two sub-clusters.