A secondary xylan-binding site enhances the catalytic activity of a single-domain family 11 glycoside hydrolase

Bacillus circulans xylanase (BcX) is a single-domain family 11 glycoside hydrolase. Using NMR-monitored titrations, we discovered that an inactive variant of this enzyme, E78Q-BcX, bound xylooligosaccharides not only within its pronounced active site (AS) cleft, but also at a distal surface region. Chemical shift perturbation mapping and affinity electrophoresis, combined with mutational studies, identified the xylan-specific secondary binding site (SBS) as a shallow groove lined by Asn, Ser, and Thr residues and with a Trp at one end. The AS and SBS bound short xylooligosaccharides with similar dissociation constants in the millimolar range. However, the on and off-rates to the SBS were at least tenfold faster than those of kon approximately 3x10(5) M(-1) s(-1) and koff approximately 1000 s(-1) measured for xylotetraose to the AS of E78Q-BcX. Consistent with their structural differences, this suggests that a conformational change in the enzyme and/or the substrate is required for association to and dissociation from the deep AS, but not the shallow SBS. In contrast to the independent binding of small xylooligosaccharides, high-affinity binding of soluble and insoluble xylan, as well as xylododecaose, occurred cooperatively to the two sites. This was evidenced by an approximately 100-fold increase in relative Kd values for these ligands upon mutation of the SBS. The SBS also enhances the activity of BcX towards soluble and insoluble xylan through a significant reduction in the Michaelis KM values for these polymeric substrates. This study provides an unexpected example of how a single domain family 11 xylanase overcomes the lack of a carbohydrate-binding module through the use of a secondary binding site to enhance substrate specificity and affinity.     

Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1

Removal of the fucose residue from the oligosaccharides attached to Asn297 of human immunoglobulin G1 (IgG1) results in a significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) via improved IgG1 binding to Fcgamma receptor IIIa. To provide structural insight into the mechanisms of affinity enhancement, we determined the crystal structure of the nonfucosylated Fc fragment and compared it with that of fucosylated Fc. The overall conformations of the fucosylated and nonfucosylated Fc fragments were similar except for hydration mode around Tyr296. Stable-isotope-assisted NMR analyses confirmed the similarity of the overall structures between fucosylated and nonfucosylated Fc fragments in solution. These data suggest that the glycoform-dependent ADCC enhancement is attributed to a subtle conformational alteration in a limited region of IgG1-Fc. Furthermore, the electron density maps revealed that the traces between Asp280 and Asn297 of our fucosylated and nonfucosylated Fc crystals were both different from that in previously reported isomorphous Fc crystals.     

Probing the structure of the Ff bacteriophage major coat protein transmembrane helix dimer by solution NMR

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively ¹⁵N-labeled peptide containing the TM segment of MCP (MCP_TM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCP_TM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.     

新城疫分离毒HN基因的分子特性和片段同源相关性

选取国内1997-2005年分离的新城疫病毒(Newcastle disease virus,NDV)24株,经蚀斑纯化克隆其血凝素-神经氨酸酶(HN)基因,与在GenBank发表的36株国内外不同时期的NDV毒株,进行氨基酸遗传变异分析,并利用SPSS8.0软件对其不同片段的氨基酸进行同源相关比较。结果显示:国内所有NDV分离毒株氨基酸高度同源,同源性为94.4%-99.4%;与LaSota、Clone30疫苗株等的氨基酸同源性为86.9%-89%;与强毒株F48E9的氨基酸同源性为87.9%-89.9%;与国外NDV的氨基酸同源性为87.2%-96.2%。系统发育分析表明:国内NDV分离毒HN遗传距离较近,而与LaSota、Clone30和F48E9遗传距离较远。国内NDV分离毒均缺乏538-540位糖基化位点。不同片段与全长的氨基酸同源性高度相关,且与前80个氨基酸相关最密切。     

A framework for power and sensitivity analyses for quantitative genetic studies of natural populations, and case studies in Soay sheep (Ovis aries)

Studies of the quantitative genetics of natural populations have contributed greatly to evolutionary biology in recent years. However, while pedigree data required are often uncertain (i.e. incomplete and partly erroneous) and limited, means to evaluate the effects of such uncertainties have not been developed. We have therefore developed a general framework for power and sensitivity analyses of such studies. We propose that researchers first generate a set of pedigree data that they wish to use in a quantitative genetic study, as well as data regarding errors that occur in that pedigree. This pedigree is then permuted using the data regarding errors to generate hypothetical 'true' and 'assumed' pedigrees that differ so as to mimic pedigree errors that might occur in the study system under consideration. Phenotypic data are then simulated across the true pedigree (according to user-defined genetic and environmental covariance structures), before being analysed with standard quantitative genetic techniques in conjunction with the 'assumed' pedigree data. To illustrate this approach, we conducted power and sensitivity analyses in a well-known study of Soay sheep (Ovis aries). We found that, although the estimation of simple genetic (co)variance structures is fairly robust to pedigree errors, some potentially serious biases were detected under more complex scenarios involving maternal effects. Power analyses also showed that this study system provides high power to detect heritabilities as low as about 0.09. Given this range of results, we suggest that such power and sensitivity analyses could greatly complement empirical studies, and we provide the computer program PEDANTICS to aid in their application.     

Behavioral syndromes and the evolution of correlated behavior in zebrafish

Studies of "behavioral syndromes" in different populations andspecies of animals can be used to shed light on the underlyingmechanisms of evolution. For example, some personality syndromessuggest the existence of an underlying hormonal link, whereasother relationships between boldness and aggression appear tobe the result of similar selective pressures. Here, we used1 wild-derived and 2 laboratory strains of zebrafish (Daniorerio) to examine relationships among 5 behavioral measures:shoaling, activity level, predator approaches, latency to feedafter a disturbance, and biting to a mirror stimulus. We foundevidence of an activity syndrome, as if underlying metaboliccosts influence variation in multiple forms of behavior. Evidencefor a relationship between boldness and aggression was alsoapparent but depended both on strain and which specific behaviorpatterns were identified as measures of "boldness." Althoughsome comparisons of laboratory and wild-derived strains wereconsistent with a domestication syndrome, others were not. Mostobserved relationships were relatively weak and occasionallyinconsistent, arguing against strong underlying genetic linkagesor pleiotropic effects relating any of the behavioral measures.Instead, it may be more important to study the details of selectivecontext or the long-term impact of linkages between some, butnot all, of a large set of genes influencing complex behavioraltraits. We found profound differences among strains in mostbehavior patterns, but few sex differences. One strain (TM1)was consistently different from the others (SH and Nadia) beingmore social, more likely to approach predators, and taking lesstime to recover from disturbance than were the other 2 strains.     

Increasing sequence correlation limits the efficiency of recombination in a multisite evolution model

The accumulation of preexisting beneficial alleles in a haploid population, under selection and infrequent recombination, and in the absence of new mutation events is studied numerically by means of detailed Monte Carlo simulations. On the one hand, we confirm our previous work, in that the accumulation rate follows modified single-site kinetics, with a timescale set by an effective selection coefficient s(eff) as shown in a previous work, and we confirm the qualitative features of the dependence of s(eff) on the population size and the recombination rate reported therein. In particular, we confirm the existence of a threshold population size below which evolution stops before the emergence of best-fit individuals. On the other hand, our simulations reveal that the population dynamics is essentially shaped by the steady accumulation of pairwise sequence correlation, causing sequence congruence in excess of what one would expect from a uniformly random distribution of alleles. By sequence congruence, we understand here the opposite of genetic distance, that is, the fraction of monomorphic sites of specified allele type in a pair of genomes (individual sequences). The effective selection coefficient changes more rapidly with the recombination rate and has a higher threshold in this parameter than found in the previous work, which neglected correlation effects. We examine this phenomenon by monitoring the time dependence of sequence correlation based on a set of sequence congruence measures and verify that it is not associated with the development of linkage disequilibrium. We also discuss applications to HIV evolution in infected individuals and potential implications for drug therapy.     

Empirical isotropic chemical shift surfaces

A list of proteins is given for which spatial structures, with a resolution better than 2.5 A, are known from entries in the Protein Data Bank (PDB) and isotropic chemical shift (ICS) values are known from the RefDB database related to the Biological Magnetic Resonance Bank (BMRB) database. The structures chosen provide, with unknown uncertainties, dihedral angles phi and psi characterizing the backbone structure of the residues. The joint use of experimental ICSs of the same residues within the proteins, again with mostly unknown uncertainties, and ab initio ICS(phi,psi) surfaces obtained for the model peptides For-(L-Ala)(n)-NH(2), with n = 1, 3, and 5, resulted in so-called empirical ICS(phi,psi) surfaces for all major nuclei of the 20 naturally occurring alpha-amino acids. Out of the many empirical surfaces determined, it is the 13C(alpha)-1H(alpha) ICS(phi,psi) surface which seems to be most promising for identifying major secondary structure types, alpha-helix, beta-strand, left-handed helix (alpha(D)), and polyproline-II. Detailed tests suggest that Ala is a good model for many naturally occurring alpha-amino acids. Two-dimensional empirical 13C(alpha)-1H(alpha) ICS(phi,psi) correlation plots, obtained so far only from computations on small peptide models, suggest the utility of the experimental information contained therein and thus they should provide useful constraints for structure determinations of proteins.