用网络方法识别生物序列motif

生物序列motif的识别是后基因组时代的一个核心问题。本文首先回顾了识别motif的几种主要算法,然后根据motif的重要性和随机性介绍了利用网络识别motif的两种具有代表性的方法：一种是建立一个随机网络混合模型,利用EM算法识别其中随机的网络motif;另一种用修正的参数流算法过滤出其中的最大密度予图,即为生物序列motif,并指出这两种方法的优劣,最后还对今后研究方向给出了讨论。     

CpG motif的免疫调节作用及机制

CpGmotif是以CpG二核苷酸为核心的,未甲基化的特殊的DNA序列,在微生物基因组中的出现频率高于在脊椎动物中的出现频率,近年来发现这种特殊的基序具有许多免疫调节作用。如刺激淋巴细胞增殖,分泌细胞因子,产生IgG2a类抗体,诱导Th1类应答,本介绍这种独特的CpGmotif的特征,可能的调节机制及应用前景。     

Sequence-dependent stability test of a left-handed β-helix motif

The left-handed β-helix (LHBH) is an intriguing, rare structural pattern in polypeptides that has been implicated in the formation of amyloid aggregates. We used accurate all-atom replica-exchange molecular dynamics (REMD) simulations to study the relative stability of diverse sequences in the LHBH conformation. Ensemble-average coordinates from REMD served as a scoring criterion to identify sequences and threadings optimally suited to the LHBH, as in a fold recognition paradigm. We examined the repeatability of our REMD simulations, finding that single simulations can be reliable to a quantifiable extent. We find expected behavior for the positive and negative control cases of a native LHBH and intrinsically disordered sequences, respectively. Polyglutamine and a designed hexapeptide repeat show remarkable affinity for the LHBH motif. A structural model for misfolded murine prion protein was also considered, and showed intermediate stability under the given conditions. Our technique is found to be an effective probe of LHBH stability, and promises to be scalable to broader studies of this and potentially other novel or rare motifs. The superstable character of the designed hexapeptide repeat suggests theoretical and experimental follow-ups.     

Cyclin-dependent kinases: a new cell cycle motif?

Increasing evidence suggests that the eukaryotic cell cycle is controlled at several checkpoints by different members of a novel class of protein kinase, the cyclin-dependent kinases. To phosphorylate their substrates, these enzymes bind to proteins of the cyclin family--proteins that are synthesized and degraded at specific points in each cell cycle. The most well known of these kinases is the 34 kDa product of the cdc2 gene in fission yeast, p34cdc2; however, several putative cyclin-dependent kinases have now been cloned or identified. Some of these closely resemble p34cdc2. Here we review these new proteins, their potential roles in the cell cycle and the cyclins with which they may interact.     

Delineation of a new structural motif involving NHN γ-turn

Macromolecules are characterized by distinctive arrangement of hydrogen bonds. Different patterns of hydrogen bonds give rise to distinct and stable structural motifs. An analysis of 4114 non-redundant protein chains reveals the existence of a three-residue, (i − 1) to (i + 1), structural motif, having two hydrogen-bonded five-membered pseudo rings (the first, an N H···OC involving the first residue, and the second being N H∙∙∙N involving the last two residues), separated by a peptide bond. There could be an additional hydrogen bond between the side-chain at (i-1) and the main-chain NH of (i + 1). The average backbone torsion angles of −76(±21)° and – 12(±17)° at i creates a tight turn in the polypeptide chain, akin to a γ-turn. Indeed, a search of three-residue fragments with restriction on the terminal C^α···C^α distance and the existence of the two pseudo rings on either side revealed the presence 14 846 cases of a variant, termed NHN γ-turn, distinct from the NHO γ-turn (2032 cases) that has traditionally been characterized by the presence of NHO hydrogen bond linking the terminal main-chain atoms. As in the latter, the newly identified γ-turns are also of two types—classical and inverse, occurring in the ratio of 1:6. The propensities of residues to occur in these turns and their secondary structural features have been enumerated. An understanding of these turns would be useful for structure prediction and loop modeling, and may serve as models to represent some of the unfolded state or disordered region in proteins.     

Structure and dynamics of human Nedd4-1 WW3 in complex with the αENaC PY motif

Nedd4-1 (neuronal precursor cell expressed developmentally downregulated gene 4-1) is an E3 ubiquitin ligase that interacts with and negatively regulates the epithelial Na⁺ channel (ENaC). The WW domains of Nedd4-1 bind to the ENaC subunits via recognition of PY motifs. Human Nedd4-1 (hNedd4-1) contains four WW domains with the third domain (WW3*) showing the strongest affinity to the PY motif. To understand the mechanism underlying this binding affinity, we have carried out NMR structural and dynamics analyses of the hNedd4-1 WW3* domain in complex with a peptide comprising the C-terminal tail of the human ENaC α-subunit. The structure reveals that the peptide interacts in a similar manner to other WW domain–ENaC peptide structures. Crucial interactions that likely provide binding affinity are the broad XP groove facilitating additional contacts between the WW3* domain and the peptide, compared to similar complexes, and the large surface area buried (83 Å²) between R430 (WW3*) and L647′ (αENaC). This corroborates the model-free analysis of the ¹⁵N backbone relaxation data, which showed that R430 is the most rigid residue in the domain (S² = 0.90 ± 0.01). Carr–Purcell–Meiboom–Gill relaxation dispersion analysis identified two different conformational exchange processes on the μs–ms time-scale. One of these processes involves residues located at the peptide binding interface, suggesting conformational exchange may play a role in peptide recognition. Thus, both structural and dynamic features of the complex appear to define the high binding affinity. The results should aid interpretation of biochemical data and modeling interfaces between Nedd4-1 and other interacting proteins.     

Pyrimidone-based series of glucokinase activators with alternative donor–acceptor motif

Glucokinase activators are a class of experimental agents under investigation as a therapy for Type 2 diabetes mellitus. An X-ray crystal structure of a modestly potent agent revealed the potential to substitute the common heterocyclic amide donor–acceptor motif for a pyridone moiety. We have successfully demonstrated that both pyridone and pyrimidone heterocycles can be used as a potent donor–acceptor substituent. Several sub-micromolar analogs that possess the desired partial activator profile were synthesized and characterized. Unfortunately, the most potent activators suffered from sub-optimal pharmacokinetic properties. Nonetheless, these donor–acceptor motifs may find utility in other glucokinase activator series or beyond.     

The ASB2β Ubiquitin-interacting motif is involved in its monoubiquitination

ASB2 proteins are E3 ubiquitin (Ub) ligases that ubiquitinate filamins. There are two ASB2 splice variants, ASB2α and ASB2β. ASB2β has a ubiquitin-binding motif (UIM) at the N-terminal region but ASB2α does not. Here, we provide the first evidence that ASB2β but not ASB2α is monoubiquitinated and that this monoubiquitination involves the UIM. Myc-tagged ASB2β and hemagglutinin (HA)-tagged Ub were co-expressed in HEK293 cells using the pCMV expression vector. Immunoprecipitation with an anti-Myc antibody followed by immunoblotting with anti-Myc and anti-HA antibodies showed an additional ASB2β protein band that had both a Myc and a HA tag. The molecular weight of this protein was larger than that of ASB2β, and the difference in molecular weight between these two proteins corresponded to the molecular weight of monoubiquitin, strongly implying that monoubiquitinated ASB2β is produced in cells. ASB2β with mutations in the UIM motif; either Glu·Asp·Glu27-29Ala·Ala·Ala mutations (ASB2β M1) or a Ser38Ala mutation, (ASB2β M2) were not monoubiquitinated, suggesting the importance of the UIM for ASB2β monoubiquitination. Furthermore, an ASB2β mutant that lacked a SOCS box (ASB2β ΔC) and did not show E3 Ub ligase activity was monoubiquitinated to the same extent as the wild-type ASB2β. In contrast, an ASB2β mutant that lacked the UIM-containing domain (ASB2β ΔN) was not monoubiquitinated. These results suggest that ASB2β but not ASB2α might be monoubiquitinated and that the ASB2β UIM motif, but not its E3 Ub ligase activity, plays a pivotal role in this monoubiquitination.     

Clathrin-binding proteins: got a motif? Join the network!

Clathrin plays a key function in membrane and protein trafficking through the endocytic and late secretory pathways. Its role as a molecular scaffold that drives formation of transport vesicles requires binding to a number of proteins with distinct functional and structural properties. Recent studies have revealed that most of these proteins interact with clathrin through surprisingly simple, linear arrangements of acidic and hydrophobic amino acid residues. This article discusses the different types of clathrin-binding proteins and motifs as well as the physiological significance of these proteins in clathrin-dependent events.     

Targeting of voltage-gated calcium channel α2δ-1 subunit to lipid rafts is independent from a GPI-anchoring motif

Voltage-gated calcium channels (Ca(v)) exist as heteromultimers comprising a pore-forming α(1) with accessory β and α(2)δ subunits which modify channel trafficking and function. We previously showed that α(2)δ-1 (and likely the other mammalian α(2)δ isoforms--α(2)δ-2, 3 and 4) is required for targeting Ca(v)s to lipid rafts, although the mechanism remains unclear. Whilst originally understood to have a classical type I transmembrane (TM) topology, recent evidence suggests the α(2)δ subunit contains a glycosylphosphatidylinositol (GPI)-anchor that mediates its association with lipid rafts. To test this notion, we have used a strategy based on the expression of chimera, where the reported GPI-anchoring sequences in the gabapentinoid-sensitive α(2)δ-1 subunit have been substituted with those of a functionally inert Type I TM-spanning protein--PIN-G. Using imaging, electrophysiology and biochemistry, we find that lipid raft association of PIN-α(2)δ is unaffected by substitution of the GPI motif with the TM domain of PIN-G. Moreover, the presence of the GPI motif alone is not sufficient for raft localisation, suggesting that upstream residues are required. GPI-anchoring is susceptible to phosphatidylinositol-phospholipase C (PI-PLC) cleavage. However, whilst raft localisation of PIN-α(2)δ is disrupted by PI-PLC treatment, this is assay-dependent and non-specific effects of PI-PLC are observed on the distribution of the endogenous raft marker, caveolin, but not flotillin. Taken together, these data are most consistent with a model where α(2)δ-1 retains its type I transmembrane topology and its targeting to lipid rafts is governed by sequences upstream of the putative GPI anchor, that promote protein-protein, rather than lipid-lipid interactions.     

Tilted peptides: a structural motif involved in protein membrane insertion?

Tilted peptides are short hydrophobic protein fragments characterized by an asymmetric distribution of their hydrophobic residues when helical. They are able to interact with a hydrophobic/hydrophilic interface (such as a lipid membrane) and to destabilize the organized system into which they insert. They were detected in viral fusion proteins and in proteins involved in different biological processes involving membrane insertion or translocation of the protein in which they are found. In this paper, we have analysed different protein domains related to membrane insertion with regard to their tilted properties. They are the N-terminal signal peptide of the filamentous haemagglutinin (FHA), a Bordetella pertussis protein secreted in high amount and the hydrophobic domain from proteins forming pores (i.e. ColIa, Bax and Bcl-2). From the predictions and the experimental approaches, we suggest that tilted peptides found in those proteins could have a more general role in the mechanism of insertion/translocation of proteins into/across membranes. For the signal sequences, they could help the protein machinery involved in protein secretion to be more active. In the case of toroidal pore formation, they could disturb the lipids, facilitating the insertion of the other more hydrophilic helices.     

Chromatin globules: a common motif of higher order chromosome structure?

Recent technological advances in the field of chromosome conformation capture are facilitating tremendous progress in the ability to map the three-dimensional (3D) organization of chromosomes at a resolution of several Kb and at the scale of complete genomes. Here we review progress in analyzing chromosome organization in human cells by building 3D models of chromatin based on comprehensive chromatin interaction datasets. We describe recent experiments that suggest that long-range interactions between active functional elements are sufficient to drive folding of local chromatin domains into compact globular states. We propose that chromatin globules are commonly formed along chromosomes, in a cell type specific pattern, as a result of frequent long-range interactions among active genes and nearby regulatory elements. Further, we speculate that increasingly longer range interactions can drive aggregation of groups of globular domains. This process would yield a compartmentalized chromosome conformation, consistent with recent observations obtained with genome-wide chromatin interaction mapping.     

Allosterism in membrane binding: a common motif of the annexins?

Annexins are a family of proteins generally described as Ca(2+)-dependent for phospholipid binding. Yet, annexins have a wide variety of binding behaviors and conformational states, some of which are lipid-dependent and Ca(2+)-independent. We present a model that captures the cation and phospholipid binding behavior of the highly conserved core of the annexins. Experimental data for annexins A4 and A5, which have short N-termini, were globally modeled to gain an understanding of how the lipid-binding affinity of the conserved protein core is modulated. Analysis of the binding behavior was achieved through use of the lanthanide Tb(3+) as a Ca(2+) analogue. Binding isotherms were determined experimentally from the quenching of the intrinsic fluorescence of annexins A4 and A5 by Tb(3+) in the presence or absence of membranes. In the presence of lipid, the affinity of annexin for cation increases, and the binding isotherms change from hyperbolic to weakly sigmoidal. This behavior was modeled by isotherms derived from microscopic binding partition functions. The change from hyperbolic to sigmoidal binding occurs because of an allosteric transition from the annexin solution state to its membrane-associated state. Protein binding to lipid bilayers renders cation binding by annexins cooperative. The two annexin states denote two affinities of the protein for cation, one in the absence and another in the presence of membrane. In the framework of this model, we discuss membrane binding as well as the influence of the N-terminus in modifying the annexin cation-binding affinity by changing the probability of the protein to undergo the postulated two-state transition.     

Is the WKS motif the tissue-factor binding site for coagulation factor VII?

Human tissue factor (TF), the membrane-bound glycoprotein receptor for the blood-clotting factor VII/VIIa, contains in its extracellular domain three repeats of the rare motif, tryptophan-lysine-serine (WKS). Murine tissue factor, which binds human factor VII/VIIa poorly, contains only one WKS motif suggesting that the WKS motif may be involved in the binding of human factor VII/VIIa to human TF. Sequence analysis has revealed a WKS motif in 23 human proteins, seven of which are involved in the coagulation process. Another five WKS-containing proteins share some functional properties with the coagulation proteins. Analysis of the properties of these proteins provides some insight into the possible functional role of the WKS motif.     

Zinc finger motif for single-stranded nucleic acids? investigations by nuclear magnetic resonance

Nuclear magnetic resonance (NMR) methods have been used to address issues regarding the relevance and feasibility of zinc binding to "zinc finger-like" sequences of the type C-X2-C-X4-H-X4-C [referred to as CCHC or retroviral-type (RT) zinc finger sequences]. One-dimensional (1D) NMR experiments with an 18-residue synthetic peptide containing the amino acid sequence of an HIV-1 RT-zinc finger domain (HIV1-F1) indicate that the sequences are capable of binding zinc tightly and stoichiometrically. 1H-113Cd spin echo difference NMR data confirm that the Cys and His amino acids are coordinated to metal in the 113Cd adduct. The 3D structure of the zinc adduct [Zn(HIV1-F1)] was determined to high atomic resolution by a new NMR-based approach that utilizes 2D-NOESY back-calculations as a measure of the consistency between the structures and the experimental data. Several interesting structural features were observed, including (1) the presence of extensive internal hydrogen bonding, and (2) the similarity of the folding of the first six residues to the folding observed by X-ray crystallography for related residues in the iron domain of rubredoxin. Structural constraints associated with conservatively substituted glycines provide further rationale for the physiological relevance of the zinc adduct. Similar NMR and structural results have been obtained for the second HIV-1 RT-zinc finger peptide, Zn(HIV1-F2). NMR studies of the zinc adduct with the NCP isolated directly from HIV-1 particles provide solid evidence that zinc finger domains are formed that are conformationally similar (if not identical) to the peptide structures.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Mrr-family nuclease motif in the single polypeptide restriction–modification enzyme LlaGI

Bioinformatic analysis of the putative nuclease domain of the single polypeptide restriction–modification enzyme LlaGI reveals amino acid motifs characteristic of the Escherichia coli methylated DNA-specific Mrr endonuclease. Using mutagenesis, we examined the role of the conserved residues in both DNA translocation and cleavage. Mutations in those residues predicted to play a role in DNA hydrolysis produced enzymes that could translocate on DNA but were either unable to cleave the polynucleotide track or had reduced nuclease activity. Cleavage by LlaGI is not targeted to methylated DNA, suggesting that the conserved motifs in the Mrr domain are a conventional sub-family of the PD-(D/E)XK superfamily of DNA nucleases.