Elucidating beta-lactamase functional mechanisms via evolutionary conservation

通过进化保守阐明β-内酰胺酶的功能机制

• 批准号: 8432993
• 负责人: Donald JACOBS
• 金额: $ 32.3万
• 依托单位: UNIVERSITY OF NORTH CAROLINA CHARLOTTE
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-15 至 2017-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8432993
• 关键词: Active Sites; Allosteric Site; Amino Acid Sequence; Antibiotic Resistance; Antibiotics; Bacteria; Bacterial Infections; Benign; Binding; Bioinformatics; Biophysics; Cell Wall; Cephalosporin Resistance; Cephalosporins; Comparative Study; Conceptions; Data; Development; Entropy; Enzymes; Equilibrium; Evolution; Family; Financial compensation; Fright; Future; Gatekeeping; Generations; Genes; Gram-Negative Bacteria; Gram-Positive Bacterial Infections; Hydrolysis; Ions; Lactamase; Lactams; Lead; Mechanics; Mediating; Metals; Methods; Metric; Modeling; Mutation; Orthologous Gene; Pattern; Penetration; Penicillin Resistance; Penicillins; Peptide Sequence Determination; Phylogenetic Analysis; Phylogeny; Property; Proteins; Public Health; Resistance; Series; Serine; Structure; Structure-Activity Relationship; System; Thermodynamics; Variant; Work; Zinc; bacterial resistance; base; beta-Lactamase; combat; comparative; design; effective therapy; enthalpy; flexibility; global health; improved; insight; interest; member; molecular dynamics; novel therapeutics; protein structure; public health relevance; resistance mechanism

DESCRIPTION (provided by applicant): Penicillins and chemically related molecules are our most abundant and common used class of antibiotics, which are characterized by a conserved 4-atom ¿-lactam ring. Historically, they have been an effective treatment to gram-positive bacterial infections; however, the cell wall of gram-negative bacteria poses an effective barrier to antibiotic penetration. Conversely, second generation cephalosporins are also effective against gram-negative bacteria because they are able to penetrate the cell wall. Nevertheless, an increasing number of bacteria are resistant due to the enzyme ?-lactamase (BL). BL, which is expressed in the cell wall, hydrolyzes the ? -lactam ring, thus rendering the antibiotic ineffective. Due to decades of antibiotic overuse, BL enzymes have alarmingly evolved additional resistances that are now breaking down our last lines of defense. For example, extended spectrum ? -lactamases (ESBL) also hydrolyze the ? -lactam ring of cephalosporins, which have generally been resistant to BL activity. As such, a better understanding of BL resistance mechanisms is imperative so that new and more effective antibiotics can be developed quickly. There are four common classes of BL enzymes, which reflect specific sequence, structure and antibiotic resistance patterns. The Class A, C and D enzymes share a serine-based hydrolysis, whereas the catalytic mechanism of Class B enzymes is based on a zinc ion. However, little is known about how dynamics and stability vary across the superfamily. Are stability and/or dynamical mechanisms conserved across the superfamily? Are certain mechanisms critical to function? Can mechanistic differences help explain antibiotic resistance patterns? Is allostery conserved across the superfamily? These are the types of unanswered questions this proposal seeks to answer. To that end, we will employ a powerful and fast computational distance constraint model (DCM) to characterize the serine-based classes. While broad characterization across the BL superfamily has not yet been done, a small number of Class A structures have been studied by NMR and molecular dynamics simulation. Interestingly, these structures appear extraordinarily rigid, punctuated by flexible loop regions that may or may not be related to function. Our preliminary DCM characterizations across Class A enzymes reproduce these results. Even so, there is significant diversity within dynamical quantities across the family, which reflects evolutionary out-groups and, in many cases, parallels the onset of extended-spectrum activities. Taken together, these preliminary results highlight how the synthesis of biophysical descriptions with the paradigm of comparative bioinformatics synergistically improves the importance and accuracy of our characterizations. As such, we propose a series of additional studies along these lines to expand our understanding of BL structure and function, potentially paving the way to new therapeutic opportunities.

描述（由申请人提供）：青霉素及其化学相关分子是我们最丰富和最常用的一类抗生素，其特征是一个保守的4原子内酰胺环。从历史上看，它们是治疗革兰氏阳性细菌感染的有效方法；然而，革兰氏阴性菌的细胞壁对抗生素的渗透形成了有效的屏障。相反，第二代头孢菌素对革兰氏阴性菌也有效，因为它们能够穿透细胞壁。然而，由于这种酶的作用，越来越多的细菌具有耐药性。内酰胺酶(提单)。在细胞壁中表达的BL能水解？-内酰胺环，从而使抗生素无效。由于几十年来抗生素的过度使用，BL酶已经惊人地进化出了额外的耐药性，现在正在打破我们最后的防线。例如，扩展频谱？-内酰胺酶（ESBL）也能水解？头孢菌素的-内酰胺环，通常对BL活性具有抗性。因此，更好地了解BL耐药机制是必要的，以便能够迅速开发新的和更有效的抗生素。有四类常见的BL酶，它们反映了特定的序列、结构和抗生素耐药模式。A类、C类和D类酶都是基于丝氨酸的水解，而B类酶的催化机制是基于锌离子的。然而，人们对整个超家族的动态和稳定性如何变化知之甚少。稳定性和/或动力机制在整个超家族中是保守的吗？某些机制对功能至关重要吗？机制差异是否有助于解释抗生素耐药性模式？变构在整个超族中守恒吗？这些都是本提案试图回答的悬而未决的问题。为此，我们将采用一个强大而快速的计算距离约束模型（DCM）来表征基于丝氨酸的类。虽然目前还没有对BL超家族进行广泛的表征，但已经通过核磁共振和分子动力学模拟研究了少量的a类结构。有趣的是，这些结构看起来非常坚硬，被可能与功能有关也可能与功能无关的柔性环区打断。我们对A类酶的初步DCM表征再现了这些结果。即便如此，在动态数量上也有显著的差异