MOLECULAR PHYSIOLOGY OF CALCIUM 
D. Martin Watterson, Ph.D., Investigator 
Eukaryotic cells respond to a diversity of physio- 
logical and pharmacological stimuli by molecular 
mechanisms that include transient rises in the intra- 
cellular concentration of ionized calcium. These in- 
tracellular calcium signals are transduced into bio- 
logical responses through the action of a class of 
calcium-binding proteins that includes calmodulin 
(CaM). Although there is a family of highly similar 
calcium-binding proteins in this class (e.g., tropo- 
nin C from skeletal muscle tissue has a number of 
physical and chemical similarities to CaM), many of 
these other calcium-binding proteins are more re- 
stricted in their tissue or phylogenetic distribution. 
CaM is ubiquitous among eukaryotes and has mul- 
tiple biological roles. Therefore it has been used as 
a prototypical example of this class of calcium-bind- 
ing proteins. 
CaM is an integral regulatory subunit of several 
enzymes, cytoskeletal structures, and membrane 
transport systems. An intriguing situation that ap- 
pears to be unique to CaM is the fact that a single 
eukaryotic cell can contain several of these CaM:en- 
zyme signal transduction complexes, and a particu- 
lar CaM:enzyme complex can be found in multiple 
tissues and cell types. Therefore, to understand 
how a eukaryotic cell is able to manage calcium sig- 
nals in a selective and quantitative manner, it is 
necessary to have a detailed understanding of the 
mechanism of action of this calcium signal trans- 
ducer, which appears to be fundamental to life and 
eukaryotic cell homeostasis. 
The goal of Dr. Watterson's laboratory is to un- 
derstand how CaM is able to transduce calcium sig- 
nals in eukaryotic cells into a specific set of biologi- 
cal responses and integrate these responses with 
other signal transduction pathways. Significant ad- 
vances have been realized during the past year as a 
result of the culmination of studies employing pro- 
tein engineering and site-specific mutagenesis, ap- 
proaches first applied to this field of research by Dr. 
Watterson's laboratory Detailed analyses of CaM 
and one CaM:enzyme complex and selected studies 
of other CaM-regulated enzymes have provided a 
generalized model of how CaM transduces a cal- 
cium signal into a biological response. This model 
proposes that there is a dynamic equilibrium 
among calcium, CaM, enzyme, and substrate, with 
the transient rise in ionized calcium that occurs 
with a cellular stimulus resulting in a perturbation 
of the equilibrium. Based on this model and an in- 
creased knowledge of the structural basis of CaM 
recognition by the enzyme, a rational basis for the 
design and production of new chimeric proteins 
has emerged, as well as a molecular model of how 
inherited mutations of genes encoding CaM or a 
CaM-regulated enzyme might bring about a selec- 
tive, nonlethal pathology. 
The group of CaM-regulated enzymes that served 
as a focal point were the protein kinases that have 
CaM as a regulatory subunit. These enzymes in- 
clude the phosphorylase kinases, the myosin light 
chain kinases (MLCK), and the type-II CaM-depen- 
dent protein kinases (CaMPK-II). Recently Dr. 
Watterson's laboratory demonstrated, through the 
combined use of computational chemistry, site-spe- 
cific mutagenesis, and enzyme regulatory activity 
analyses of CaM, that, in addition to hydrophobic 
interactions, certain charge features in both halves 
of the CaM molecule are critically important for the 
activation of CaM-regulated protein kinases. Fur- 
thermore, these charge properties of CaM appear 
to provide an element of selectivity to CaM's inter- 
action with various enzymes, especially among pro- 
tein kinases. For example, there is a selective pref- 
erence by MLCK for the presence of a carboxylate 
function at residues 84 and 120 in two different a- 
helices of CaM that flank a recessed hydrophobic 
surface in the carboxyl-terminal half of CaM. In 
contrast, the activity of CaM:CaMPK-II complexes is 
slightly perturbed by changes at residue 120 of 
CaM (carboxyl-terminal domain) but not by 
changes at residue 84 of CaM (central helix). Con- 
sistent with the differences in regulatory activity, 
the ability of MLCK or CaMPK-II to bind CaM was 
selectively perturbed by mutations in these regions 
of CaM. 
The approach in these studies was based on the 
concept of perturbation mutagenesis screening and 
chemical complementarity in macromolecular rec- 
ognition. The key observations that formed the 
foundation of the studies were 1) the presence in 
CaM of amphiphilic helices with an asymmetric dis- 
tribution of negative-charge properties, 2) the abil- 
ity to perturb selectively the activity of CaM:enzyme 
complexes when helical regions of CaM are per- 
turbed by charge-reversal site-specific mutagenesis, 
and 3) the presence of positive-charge clusters in 
peptide fragments of MLCK that bind CaM with an 
affinity and selectivity approximating that of the na- 
tive enzyme. The distance between Glu-84 and Glu- 
Continued 
123 
