The SI 00 family of proteins are involved in many physiological processes. Aberrant levels ofthese signaling molecules have also been associated with various forms of cancer. Some S100 proteins have been found to directly promote the advancement of certain tumors via protein-protein interactions. The long- term objective ofthis proposal is to study the calcium-dependent interaction between S100 proteins and their molecular targets. A majority of SI 00 members bind calcium with a low binding affinity, unless a target is present. However, S100A5, an SI00 protein associated with recurring meningiomas and some astrocytomas, binds calcium with an affinity 10-100 times greater than any other SI 00 protein, independent of target. Determining the source of SI OOAS's high affinity for calcium in the absence of target can give us insight into how these molecules interact with targets in physiological and pathophysiological processes (ie. tumor progression). We have preliminary evidence to suggest that S100A5 lacks the protein dynamics common to the SI 00 family which increases its affinity for calcium. The goal of Aim 1 is to solve the structure of calcium-Si 00A5 with X-ray crystallography. Of particular interest is the calcium coordination in SI OOAS's canonical EF-hand which may be unlike most SI 00 proteins. An extra ligand at the ninth position ofthe calcium coordinating sphere, much like the protein parvalbumin, may potentially enhance calcium binding and we intend to determine this directly with X-ray crystallography.
In aim 2, we will solve the structure of S100A5 bound to a peptide derived from the intracellular regulatory loop ofthe sodium-calcium channel (NCX) using NMR. An SIOOAS-target complex structure can give important information regarding general dynamic characteristics ofthe complex from multiple NMR parameters, such as NOEs or chemical exchange (ex. Rex). The SIOOAS-peptide structure also has biological significance in neurons. We predict that S100A5 plays a role in calcium homeostasis by directly competing with CaM to bind NCX thus inhibiting calcium efflux.
Our final aim i s to compare the protein dynamics of S100A5 +/- target to known low (ex. SIOOB-calcium) and high (ex. SIOOB-calcium+target) calcium affinity states. Through the use of specific 15N and 2H NMR relaxation measurements, we can directly ascertain the dynamics of backbone and side chain amide groups and methyl-bearing side chains on a variety of time scales, from ms to ps. Since SI 00 proteins are elevated in multiple disease states, comprehension ofthe molecular interactions between SI 00 proteins and targets can provide objectives for novel therapeutics, such as the ongoing drug design program in the Weber lab for inhibiting S100B as a means to treat malignant melanoma.