Human carbonic anhydrase IX (hCA IX) expression in many cancers is associated with hypoxic tumors and poor patient outcome. Inhibitors of hCA IX have been used as anticancer agents with some entering Phase I clinical trials. hCA IX is transmembrane protein whose catalytic domain faces the extracellular tumor milieu, which is typically associated with an acidic microenvironment. Here, we show that the catalytic domain of hCA IX (hCA IX-c) exhibits the necessary biochemical and biophysical properties that allow for low pH stability and activity. Furthermore, the unfolding process of hCA IX-c appears to be reversible, and its catalytic efficiency is thought to be correlated directly with its stability between pH 3.0 and 8.0 but not above pH 8.0. To rationalize this, we determined the X-ray crystal structure of hCA IX-c to 1.6 Å resolution. Insights from this study suggest an understanding of hCA IX-c stability and activity in low-pH tumor microenvironments and may be applicable to determining pH-related effects on enzymes.

The advances of serial crystallography techniques at synchrotron and X-ray free electron laser facilities have made possible the acquisition of useable data sets to determine 3-dimensional structures of macromolecules from micro- to nanosized crystals. In addition, the same technological hallmarks have contributed significantly to the field of time-resolved crystallography. However, the production of usable crystalline slurries for serial crystallographic experiments has been one of the limiting factors and contributes to an alternative sample “bottleneck” in crystal growth. In this study, we propose a method: labeled microbatch mixing (MBM), which has the capability to produce large quantities of microcrystals of macromolecules suitable for serial crystallographic experiments. This is shown to be successful for producing lysozyme, carbonic anhydrase, and adeno-associated virus crystals. MBM takes advantage of secondary nucleation induced by mixing via the application of steady agitation during the crystallization process. This leads to excessive nucleation, resulting in large quantities of well-diffracting microcrystals. MBM therefore presents a method that can potentially be applied to a range of macromolecules and a possible simple protocol to produce microcrystals for serial crystallographic experiments.

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