Calia Cianna N, Paesani Francesco
Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, California 92093, United States.
Materials Science and Engineering, University of California San Diego, La Jolla, California 92093, United States.
J Phys Chem B. 2025 Sep 18;129(37):9357-9372. doi: 10.1021/acs.jpcb.5c05027. Epub 2025 Sep 3.
Cold-adapted organisms frequently express antifreeze proteins (AFPs) that facilitate their survival at low temperatures, with some especially potent insect AFPs exhibiting β-solenoid structures with ice-binding threonine ladders. β-solenoids exist in nature in numerous forms and emerging protein design technologies may afford opportunities to diversify them further, suggesting the possibility of developing a variety of new AFPs by installing a threonine ladder on non-AFP natural or designed β-solenoids. However, early attempts at such engineering, combined with differences observed between AFPs and structurally similar ice-nucleating proteins, have raised a critical question: Will a threonine ladder show essentially the same behavior regardless of the β-solenoid scaffold that hosts it, or does the specific solenoid scaffold significantly affect a threonine ladder's structural characteristics (and thus potentially alter its suitability for ice binding)? We set out to address this question by creating distinct variants of a simplified model β-solenoid for analysis structure prediction and molecular dynamics simulations. Our findings indicate that local structural details such as the distance between the hydroxyl groups of adjacent threonines in a TXT motif can vary depending on the β-solenoid scaffold. In the most extreme example among our model solenoids, we observed in simulations that differences in only inward-facing residues of the scaffold were sufficient to influence the presence of ordered channel waters between the threonines, a noted feature of natural ice-binding threonine surfaces such as that of TmAFP. While additional studies will be necessary to expand on how such distinctions affect activity, these results emphasize that the impact of a particular β-solenoid scaffold on the local geometry of a threonine ladder may be a pertinent consideration in future efforts to design novel hyperactive AFPs to support applications ranging from biomedical cryopreservation to food science. We conclude our present investigation with a preliminary exploration of how this and other considerations manifest in a proposed workflow for generating predicted AFP-like β-solenoids using AlphaFold and ProteinMPNN.
冷适应生物经常表达抗冻蛋白（AFP），这些抗冻蛋白有助于它们在低温下生存，一些特别有效的昆虫抗冻蛋白呈现出具有冰结合苏氨酸梯的β-螺线管结构。β-螺线管在自然界中以多种形式存在，新兴的蛋白质设计技术可能为进一步使其多样化提供机会，这表明通过在非AFP天然或设计的β-螺线管上安装苏氨酸梯来开发多种新型AFP的可能性。然而，早期的此类工程尝试，再加上在AFP和结构相似的冰核蛋白之间观察到的差异，引发了一个关键问题：无论承载它的β-螺线管支架如何，苏氨酸梯是否会表现出基本相同的行为，或者特定的螺线管支架是否会显著影响苏氨酸梯的结构特征（从而可能改变其与冰结合的适宜性）？我们着手通过创建一个简化模型β-螺线管的不同变体来解决这个问题，用于结构预测和分子动力学模拟分析。我们的研究结果表明，诸如TXT基序中相邻苏氨酸羟基之间的距离等局部结构细节可能会因β-螺线管支架而异。在我们的模型螺线管中最极端的例子中，我们在模拟中观察到，仅支架向内的残基差异就足以影响苏氨酸之间有序通道水的存在，这是天然冰结合苏氨酸表面（如TmAFP）的一个显著特征。虽然需要进一步的研究来扩展此类差异如何影响活性，但这些结果强调，在未来设计新型高活性AFP以支持从生物医学冷冻保存到食品科学等应用的努力中，特定β-螺线管支架对苏氨酸梯局部几何形状的影响可能是一个相关的考虑因素。我们通过初步探索在使用AlphaFold和ProteinMPNN生成预测的AFP样β-螺线管的拟议工作流程中，这一考虑因素和其他考虑因素是如何体现的，来结束我们目前的研究。
