Departamento de Cristalografía y Biología Estructural, Instituto de Química-Física "Rocasolano," Consejo Superior de Investigaciones Cientificas, 28006 Madrid, Spain.
Proc Natl Acad Sci U S A. 2013 Oct 15;110(42):16808-13. doi: 10.1073/pnas.1300118110. Epub 2013 Oct 1.
The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the DD-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain--a remarkable 60 Å distant from the DD-transpeptidase active site--discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design.
青霉素结合蛋白 2a（PBP2a）的表达是耐甲氧西林金黄色葡萄球菌（MRSA）对β-内酰胺类抗生素广泛临床耐药的基础。细菌的高分子量青霉素结合蛋白在不同的结构域中催化转糖基酶和转肽酶活性，这些活性是合成构成细菌细胞壁的肽聚糖聚合物所必需的。在对β-内酰胺类抗生素敏感的细菌中，其青霉素结合蛋白（PBPs）的转肽酶活性由于β-内酰胺类抗生素对活性位点丝氨酸的不可逆酰化而丧失。相比之下，MRSA 的 PBP2a 对β-内酰胺酰化具有抗性，并成功催化完成细胞壁所需的 DD-转肽反应。无法用β-内酰胺类抗生素控制 MRSA 感染仍然是一个持续存在的公共卫生问题。我们在此报告了一个变构结合域的鉴定——通过对可溶性 PBP2a 构建体的晶体学分析发现，这个变构结合域距离 DD-转肽酶活性位点有 60Å 的距离。当这个变构位点被占据时，一个多残基构象变化导致活性位点打开，从而允许底物进入。同样的晶体学分析还揭示了三个变构配体的身份：Muramic 酸（肽聚糖的糖成分）、细胞壁肽聚糖和头孢洛林，这是一种最近批准的抗 MRSAβ-内酰胺类抗生素。抗 MRSAβ-内酰胺类抗生素能够刺激活性位点的变构开放，从而使 PBP2a 易于被第二个β-内酰胺分子失活，这为β-内酰胺类抗生素基于结构的设计开辟了一个前所未有的领域。
