NMR solution structure of a cyanovirin homolog from wheat head blight fungus

Members of the cyanovirin-N homolog (CVNH) lectin family are found in bacteria, fungi and plants. As part of our ongoing work on CVNH structure-function studies, we determined the high-resolution NMR solution structure of the homolog from the wheat head blight disease causing ascomycetous fungus Gibberella zeae (or Fusarium graminearum), hereafter called GzCVNH. Like cyanovirin-N (CV-N), GzCVNH comprises two tandem sequence repeats and the protein sequence exhibits 30% identity with CV-N. The overall structure is similar to those of other members of the CVNH family, with the conserved pseudo-symmetric halves of the structure, domains A and B, closely resembling recently determined structures of Tuber borchii, Neurospora crassa, and Ceratopteris richardii CVNH proteins. Although GzCVNH exhibits a similar glycan recognition profile to CV-N and specifically binds to Manα(1-2)Manα, its weak carbohydrate binding affinity to only one binding site is insufficient for conferring anti-HIV activity.     

A designed chimeric cyanovirin‐N homolog lectin: Structure and molecular basis of sucrose binding

The NMR and X‐ray structures of a designed chimeric cyanovirin‐N homolog (CVNH) protein were determined. The individual halves of the structure are similar to their counterparts in the parent proteins, with domains A and B resembling the structures of TbCVNH and NcCVNH, respectively. No significant differences between the solution and crystal conformations were observed, although details in loop conformations and distinct crystal packing‐induced features are present. Carbohydrate binding studies by NMR revealed affinity and specificity for Glcα(1‐2)Frc and Manα(1‐2)Man, and the parental half that is devoid of any sucrose affinity in NcCVNH was transformed into a genuine sucrose binding site in the context of the chimera. The atomic details of sugar recognition are seen in the crystal structure of the protein with two bound Glcα(1‐2)Frc molecules. Both sugars exhibit different conformations around the glycosidic bond and engage in unique hydrogen bonding networks in the two sites. Although the protein is able to bind two Manα(1‐2)Man molecules, a property associated with HIV‐inactivation, no anti‐HIV activity was observed for the hybrid protein. These results provide the structural basis for sugar recognition in the CVNH family and aid in deciphering the relationship between sugar binding and anti‐HIV activity. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

CVNH结构域的进化分析和选择压力检测

蓝藻抗病毒蛋白-N(Cyanovirin-N,CV-N)具有广谱抗病毒活性,其同源物构成CVNH(Cyanovirin-N homology)蛋白家族,并且家族成员的抗人类免疫缺陷病毒结构域在进化上非常保守。文章通过重建基因树对CVNH结构域的"零散分布"特点作了更为细致的了解,发现在黑曲霉、费氏曲菌、产黄青霉、粗糙脉孢霉、蓝杆藻和水蕨等物种中存在多份该结构域拷贝。在此基础上,分别采用机理式模型(Mechanistic model)和MEC模型(Mechanistic-empirical combination model)对CVNH结构域序列位点进行适应性进化分析,结果显示:1)两类模型均未检测到统计上显著的正选择位点;2)净化选择对CVNH起主导作用;3)MEC模型更适合所研究的数据。进一步使用"支-特异"模型和"支-位点"模型对蓝杆菌菌株7822和7424的祖先分支进行检测,发现该分支经历过适应性进化,并且鉴定出6个正选择位点(34L、63L、13H、76C、78K和80I)。     

CVNH 结构域进化分析及选择压力检测

蓝藻抗病毒蛋白-N(Cyanovirin-N,CV-N)具有广谱抗病毒活性;其同源物构成 CVNH 蛋白家族,并且家族成员的抗人类免疫缺陷病毒结构域在进化上非常保守。本研究通过重建基因树对 CVNH 结构域的“零散分布”特点作了更为细致的了解,发现在黑曲霉、费氏曲菌、产黄青霉、粗糙脉孢霉、蓝杆藻和水蕨等物种中该结构域存在多份拷贝。在此基础上,分别采用机理式模型和 MEC 模型对 CVNH 结构域序列位点进行适应性进化分析,结果显示：（1）两类模型均未检测到统计上显著的正选择位点;（2）净化选择对 CVNH 起主导作用;（3）MEC 模型更适合所研究数据。进一步使用“支-特异”模型和“支-位点”模型对蓝杆菌菌株7822和7424的祖先分支进行检测,发现该分支经历过适应性进化,并且鉴定出6 个正选择位点(34L、63L、13H、76C、78K 和 80I)。这些结果对后续的 CVNH 功能验证和借助基因工程手段改良蛋白的抗病毒活性具重要意义。