pH-Dependent Binding and Releasing Mechanism of Acetate in the Inner Water Cavity of Heliorhodopsin

The high-resolution structure of heliorhodopsin crystallized
at
low pH reveals the presence of a planar triangle molecule, acetate,
in the inner water cavity. Here, we investigate how the acetate molecule
is stabilized at the counterion Glu107 moiety, using molecular dynamics
(MD) simulations and a quantum mechanical/molecular mechanical (QM/MM)
approach. QM/MM calculations indicate that the density is best described
as acetate among triangle acids, including nitric acid and bicarbonate.
The calculated protonation state indicates that protonated acetate
donates an H-bond to deprotonated Glu107 in the low-pH crystal structure.
The observed red-shift of ∼30 nm in the absorption wavelength
with pKa ≈ 4 is likely due to the
His23/His80 protonation, rather than the Glu107 protonation. MD simulations
also show that acetate can exist at the Glu107 moiety only when it
is protonated. When ionized, acetate is released from the Glu107 moiety
via Asn101 at the channel bottleneck and Arg91 on the intracellular
protein surface. These observations could explain how acetate binds
at low pH and releases at high pH.

在低pH条件下结晶的螺旋视紫红质（heliorhodopsin）的高分辨率结构显示，其内部水腔中存在平面三角形分子乙酸盐。本研究采用分子动力学（MD）模拟与量子力学/分子力学（QM/MM）联用方法，探究了乙酸盐分子在抗衡离子Glu107（谷氨酸107）位点的稳定机制。QM/MM计算结果表明，在包括硝酸、碳酸氢根在内的三角形酸类中，该电子密度特征最符合乙酸盐的匹配度。计算得到的质子化状态显示，在低pH晶体结构中，质子化乙酸盐可作为氢键供体，与去质子化的Glu107形成氢键。观测到的吸收波长红移约30 nm、pKa≈4的现象，大概率由His23（组氨酸23）与His80（组氨酸80）的质子化状态变化所导致，而非Glu107的质子化改变。MD模拟还证实，仅当乙酸基质子化时，它才能稳定驻留于Glu107位点；当乙酸盐发生电离后，会通过通道瓶颈处的Asn101（天冬酰胺101）与细胞内蛋白表面的Arg91（精氨酸91），从Glu107位点释放出来。上述研究结果可合理解释乙酸盐在低pH条件下结合、高pH条件下释放的分子机制。