SAR by TR-HT-SAXS

Well that’s an acronym soup! SAR by NMR was the first practical fragment-finding method, and over the years Practical Fragments has covered lots of other techniques. Small-angle X-ray scattering, or SAXS, has not been among them. As the name suggests, this technique uses X-rays, typically produced at a synchrotron. However, unlike conventional crystallography, it doesn’t require crystalline material. Instead, proteins in solution are analyzed to provide information on their size and shape. The resolution is too low to assess small molecule binding, but suitable for observing dimerization or changes in conformation.

 

Time-resolved SAXS, or TR-SAXS, examines SAXS over time in response to a trigger. For example, you can rapidly add a ligand to a protein and watch for changes in conformation. And HT simply means high throughput. A recent Nature Chemical Biology paper from Chris Brosey, John Tainer, and collaborators at the University of Texas MD Anderson Center, Lawrence Berkeley National Laboratory, University of California Santa Cruz, and University of Arkansas for Medical Sciences Little Rock describes structure-activity relationships by time-resolved high throughput small-angle X-ray scattering (TR-HT-SAXS).

 

The researchers were interested in apoptosis-inducing factor (AIF), a mitochondrial protein with potential implications for cancer and other diseases. AIF normally exists as a monomer in complex with an FAD cofactor. Binding of NADH causes reduction of FAD to FADH^- and concomitant dimerization of the protein. Could fragments do the same, allowing dimerization on demand?

 

A library of 2500 fragments purchased from Life Chemicals was screened at 0.75-1.5 mM against the AIF-FAD complex using differential scanning fluorimetry (DSF), and those that raised or lowered the temperature by more than 1.7 ºC were further characterized by microscale thermophoresis (MST). This led to 32 binders and 7 negative controls, or molecules that did not confirm either by DSF or MST. (Side note: although many people discount compounds that give negative thermal shifts, the natural ligand NADH lowers the melting temperature of AIF by a whopping 10.8 ºC.)

 

Next, the fragment binders and negative controls were screened at 0.5-1 mM by TR-SAXS. Intense X-rays cause reduction of the FAD cofactor, but in the absence of NADH or other ligands the AIF protein remains monomeric. However, some fragments did cause dimerization of the protein during TR-SAXS. Interestingly, these fragments were structurally related to one another. Subsequent crystallography revealed that they bind where NADH normally binds and make some of the same interactions to induce protein dimerization. The paper includes much more detailed characterization, including mutagenesis, spectroscopic, and protein crosslinking experiments to further understand the mechanism.

 

TR-SAXS is an interesting addition to our toolbox of biophysical methods suitable for fragment screening. It does have some disadvantages, such as the need for large amounts of protein at high concentrations: 67 µM in this case. Also, the “HT” may be somewhat aspirational, with a current throughput of 100-200 compounds per synchrotron shift. Finally, the technique is probably best suited to well-characterized proteins where SAXS data can be carefully modeled. With these limitations in mind, it will be fun to see how generally TR-SAXS finds fragments that alter the conformation and multimerization of proteins.