Rational Design of New Binding Specificity by Simultaneous Mutagenesis of Calmodulin and a Target Peptide

Abstract

Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies CaM's role as a shared participant in calciumdependent signal transduction pathways, but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/ target pairs that retain high affinity for one another, but lack affinity for wild-type CaM and its natural interaction partners, would therefore be useful as sensor components, and possibly also as elements of "synthetic" cellular signaling networks. Here we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal muscle light chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins, and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple linked binding equilibria. Figure 1A). (2,3) Calcium-loaded CaM binds to isolated target sequences with affinity comparable to its complexes with the intact proteins, and with dissociation constants often in the nanomolar range. With few exceptions, CaM ligands tend to be highly basic, presenting lysine and arginine residues that form salt-bridge networks with negatively-charged and polar amino-acid side chains bordering the binding cleft on CaM ( Figure 1B). In addition, many CaM targets appear to be anchored by a key pair of bulky groups spaced apart by 2.5 or 3.5 helical turns. (4) Deletion or mutation of these anchor residues dramatically reduces CaMbinding affinity. (5,6) Considerable interest in CaM/peptide interactions has revolved around potential applications in biotechnology. CaM-affinity chromatography (7) The ability to understand and control determinants of binding specificity at the CaM/target interface is important both for biotechnological applications and for appreciation of how CaM transduces metabolic signals via multiple protein interaction networks. Many studies have probed the contributions of both the charged and hydrophobic anchor residues to CaM binding affinity, (5,6,(19)(20)(21)(22)) but relatively few have sought to manipulate specificity through purposeful mutagenesis of one or both binding partners. A step in this direction was taken by Mayo and colleagues,(23,24) and involved the integration of computational and experimental methods to bias CaM spe...

Calcium-sensitive MRI contrast agents based on superparamagnetic iron oxide nanoparticles and calmodulin

“…The solid curve (filled circles) represents data from a 3:1 binary sensor (3X1:1M) made from conjugates with M13 and XCaM E104Q (13), a CaM mutant with broadened calcium sensitivity (EC 50 of 0.8 Ϯ 0.3 M, n ϭ 3). The dashed curve (open circles) represents data from an analogous sensor (3X2:1M) formed with an XCaM mutant (S81R͞E83K͞E87R͞E104Q) further modified to reduce its affinity for the M13 peptide (25). Because protein complex formation and calcium binding are linked processes, this variant also exhibits lower calcium affinity (EC 50 of 10 Ϯ 2 M, n ϭ 2) than 3X1:1M.…”

“…The DLS and MRI data from variant sensors, therefore, show collectively that the appropriate incorporation of protein mutations can alter either the cooperativity or the EC 50 of calcium binding, optimizing the sensor for reporting in different Ca 2ϩ concentration ranges. Mutagenesis of the sensor components may also alter the sensor's propensity to cross-react with endogenous CaM-related proteins in vivo (25).…”

“…Endogenous CaM is expected to pose the greatest ''threat'' in this regard because its intracellular concentration can sometimes be Ͼ1 M (31-33). To address this problem, our laboratory has adopted a mutational approach similar to one used recently to modify fluorescent CaM-based calcium sensors (25,34). Second, although the sensors will be targeted to cell cytoplasms, it is possible that some may be trapped in endosomes or other compartments where they cannot function properly.…”

Challenges in the computational design of proteins

“…Their goal was to obtain a pair: designed protein-mutated DNA molecules were able to interact among themselves, but the designed protein did not interact with wild-type DNA and natural endonuclease was not able to cut the mutated DNA. This and other examples (Green et al 2006;Joachimiak et al 2006) show the usefulness of introducing negative design when considering specificity. Fortunately, affinity is linked to specificity and sometimes, when optimizing for affinity, specificity is found without the need to introduce negative design (Looger et al 2003;Cochran et al 2005;Hu et al 2008;Yosef et al 2009).…”

Progress in computational protein design

“…Sampling known variation in rigid-body binding orientation likely contributed to the specificity, and despite the creation of a novel hydrogen-bond network, the majority of the specificity switch was due to a single hydrophobic mutation. In addition, explicit negative design was used by Baker and colleagues to successfully redesign the specificity of an endonuclease [31**], by Keating and colleagues to convert a homotetramer into a heterotetramer [32], and by Jasanoff, Tidor, and their colleagues to alter calmodulin specificity [33]. These data together support the need for elements of negative design for creating protein-protein interaction specificity.…”