Exploring the origins of binding specificity through the computational redesign of calmodulin.

Abstract

Calmodulin (CaM) is a second messenger protein that has evolved to bind tightly to a variety of targets and, as such, exhibits low binding specificity. We redesigned CaM by using a computational protein design algorithm to improve its binding specificity for one of its targets, smooth muscle myosin light chain kinase (smMLCK). Residues in or near the CaM/smMLCK binding interface were optimized; CaM interactions with alternative targets were not directly considered in the optimization. The predicted CaM sequences were constructed and tested for binding to a set of eight targets including smMLCK. The best CaM variant, obtained from a calculation that emphasized intermolecular interactions, showed up to a 155-fold increase in binding specificity. The increase in binding specificity was not due to improved binding to smMLCK, but due to decreased binding to the alternative targets. This finding is consistent with the fact that the sequence of wild-type CaM is nearly optimal for interactions with numerous targets.

Submission Details

ID: 9njU6qMX

Submitter: Marie Ary

Submission Date: Oct. 31, 2018, 3:40 p.m.

Version: 1

Publication Details
Shifman JM;Mayo SL,Proc Natl Acad Sci U S A (2003) Exploring the origins of binding specificity through the computational redesign of calmodulin. PMID:14597710
Additional Information

Study Summary

Number of data points 97
Proteins Calmodulin-1
Unique complexes 30
Assays/Quantities/Protocols Experimental Assay: Kd, circular dichroism ; Experimental Assay: Kd, fluorescence at 318 ; Derived Quantity: Specificity increase ; Derived Quantity: Relative binding (Kd_var/Kd_WT) ; Computational Protocol: Energy
Libraries Kd, relative Kd, and specificity increase of calmodulin variants to CaMKK (Table 2) ; Kd, relative Kd, and specificity increase of calmodulin variants to CaMKII (Table 2) ; Kd, relative Kd, and specificity increase of calmodulin variants to CaMKI (Table 2) ; Kd, relative Kd, and specificity increase of calmodulin variants to peptide1 (Table 2) ; Kd, relative Kd, and specificity increase of calmodulin variants to melittin (Table 2) ; Kd, relative Kd, and specificity increase of calmodulin variants to spectrin (Table 2) ; Kd, relative Kd, and specificity increase of calmodulin variants to skMLCK (Table 2) ; Kd, relative Kd, specificity increase, and energy of calmodulin variants to smMLCK (Tables 1-4)

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
90.5 Calmodulin-1 Q6R520 CALM_OREMO
96.5 Calmodulin-1 P62146 CALMA_ARBPU
93.2 Calmodulin-1 Q9GRJ1 CALM_LUMRU
98.5 Calmodulin-1 P62150 CALM_ORYLA
100.0 Calmodulin-1 O96081 CALMB_HALRO
98.5 Calmodulin-1 O97341 CALM_SUBDO
94.2 Calmodulin-1 P62203 CALM_PLAF7
94.2 Calmodulin-1 P24044 CALM_PLAFA
97.0 Calmodulin-1 Q95NI4 CALM_HALOK
92.9 Calmodulin-1 P41041 CALM_PNECA
91.2 Calmodulin-1 P0DP23 CALM1_HUMAN
91.2 Calmodulin-1 P0DP26 CALM1_MOUSE
91.2 Calmodulin-1 P0DP29 CALM1_RAT
91.2 Calmodulin-1 P0DP33 CALM1_XENLA
91.2 Calmodulin-1 Q9UB37 CALM2_BRALA
91.2 Calmodulin-1 P0DP24 CALM2_HUMAN
91.2 Calmodulin-1 P0DP27 CALM2_MOUSE
91.2 Calmodulin-1 P0DP30 CALM2_RAT
91.2 Calmodulin-1 P0DP25 CALM3_HUMAN
91.2 Calmodulin-1 P0DP28 CALM3_MOUSE
91.2 Calmodulin-1 P0DP31 CALM3_RAT
91.2 Calmodulin-1 P62144 CALM_ANAPL
91.2 Calmodulin-1 P62157 CALM_BOVIN
91.2 Calmodulin-1 P62149 CALM_CHICK
91.2 Calmodulin-1 Q6IT78 CALM_CTEID
91.2 Calmodulin-1 Q6PI52 CALM_DANRE
91.2 Calmodulin-1 P62156 CALM_ONCSP
91.2 Calmodulin-1 Q71UH6 CALM_PERFV
91.2 Calmodulin-1 Q5RAD2 CALM_PONAB
91.2 Calmodulin-1 P62160 CALM_RABIT
91.2 Calmodulin-1 Q6YNX6 CALM_SHEEP
91.2 Calmodulin-1 P21251 CALM_STIJA
91.2 Calmodulin-1 P62151 CALM_TETCF
91.2 Calmodulin-1 P0DP34 CAM2A_XENLA
91.2 Calmodulin-1 P0DP35 CAM2B_XENLA
100.0 Calmodulin-1 P05932 CALMB_ARBPU
96.5 Calmodulin-1 Q9HFY6 CALM_BLAEM
100.0 Calmodulin-1 P02594 CALM_ELEEL
94.2 Calmodulin-1 P11118 CALM_EUGGR
100.0 Calmodulin-1 P02595 CALM_PATSP
100.0 Calmodulin-1 P11121 CALM_PYUSP
100.0 Calmodulin-1 P62184 CALM_RENRE
93.5 Calmodulin-1 P69097 CALM_TRYBB
93.5 Calmodulin-1 P69098 CALM_TRYBG
93.5 Calmodulin-1 P18061 CALM_TRYCR