Knappe Daniel, Goldbach Tina, Hatfield Marcus P D, Palermo Nicholas Y, Weinert Stefanie, Sträter Norbert, Hoffmann Ralf, Lovas Sándor
Institute für Bioanalytische Chemie, Biotechnologisch-Biomedizinisches Zentrum, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany.
Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, USA.
Protein Pept Lett. 2016;23(12):1061-1071. doi: 10.2174/0929866523666160719124712.
The bacterial protein DnaK promotes folding of newly synthesized polypeptide chains, refolding of misfolded proteins, and protein trafficking. Assisted refolding is especially important under stress conditions induced by antibiotic therapies reducing the desired bactericidal effects. DnaK is supposedly targeted by proline-rich antimicrobial peptides (PrAMPs), but Escherichia coli ΔdnaK mutants and wild type strains are equally susceptible indicating further intracellular targets, such as the 70S ribosome. Crystal structures of PrAMPDnaK- complexes revealed forward and reverse binding modes at the substrate binding domain. Here, we used these ligand-target structures for the first time to rationally optimize peptides using molecular modeling and docking leading to the prediction of four-residue long sequences for improved binding to DnaK. When these sequences were used to replace the original sequence stretch in Onc72, most peptides showed significantly reduced dissociation constants (Kd) determined by fluorescence polarization. In a second approach, the X-ray structures of Api88 and Onc72 bound to DnaK were examined to predict substitutions prone to stronger interactions. Among the 36 peptides obtained from both approaches, six derivatives bound to DnaK with more than 10-fold higher affinities (Kd values in the low micromolar to nanomolar range). Peptides binding stronger to DnaK showed the same minimal inhibitory concentrations against wild type E. coli as the original peptide, but were slightly less active for ΔdnaK mutants. However, one peptide was able to overcome the resistance in an E. coli mutant lacking the SbmA transporter obligatory for the uptake of PrAMPs including Api88 and Onc72. Thus, it´s tempting to speculate that DnaK might be involved in the translocation of PrAMPs into E. coli.
细菌蛋白DnaK可促进新合成多肽链的折叠、错误折叠蛋白的重新折叠以及蛋白质运输。在抗生素治疗诱导的应激条件下，辅助重新折叠尤为重要，因为这会降低所需的杀菌效果。据推测，富含脯氨酸的抗菌肽（PrAMPs）靶向DnaK，但大肠杆菌ΔdnaK突变体和野生型菌株同样易感，这表明存在其他细胞内靶点，如70S核糖体。PrAMPDnaK复合物的晶体结构揭示了在底物结合域的正向和反向结合模式。在这里，我们首次使用这些配体-靶点结构，通过分子建模和对接合理优化肽段，从而预测出四个残基长的序列，以改善与DnaK的结合。当这些序列用于替换Onc72中的原始序列片段时，大多数肽段通过荧光偏振测定显示出显著降低的解离常数（Kd）。在第二种方法中，研究了与DnaK结合的Api88和Onc72的X射线结构，以预测易于产生更强相互作用的取代。从这两种方法获得的36种肽段中，有六种衍生物与DnaK的结合亲和力提高了10倍以上（Kd值在低微摩尔到纳摩尔范围内）。与DnaK结合更强的肽段对野生型大肠杆菌的最小抑菌浓度与原始肽段相同，但对ΔdnaK突变体的活性略低。然而，有一种肽能够克服大肠杆菌突变体中的耐药性，该突变体缺乏摄取包括Api88和Onc72在内的PrAMPs所必需的SbmA转运蛋白。因此，很容易推测DnaK可能参与PrAMPs转运到大肠杆菌中。
