Tuning Ca 2+ binding affinities by rational design

Understanding the key determinants for calcium binding is essential for designing calcium sensors with different affinities. According to the study of putative calcium binding sites fea- tured by pentagonal bipyramidal geometry structure(42), two factors can influence the calcium binding affinity, 1) the coordination number of the binding ligands, 2) the geometric arrange- Figure 1.6 The SR calcium release triggered by excitation-contraction coupling

(E-C coupling) of skeletal muscle. Excitation-contraction coupling of skeletal muscle. A muscle fiber is excited via the nerve by an endplate potential and generates an action potential, which spreads out along the surface membrane and the transverse tubular system into the deeper parts of the muscle fiber. The dihydropyridine (DHP) receptor senses the membrane depolarization, alters its conformation, and activates the ryanodine receptor, which releases Ca2+ _from the SR, a Ca2+ _{store. Ca}2+ _{binds to troponin and activates the so-called contrac-} tile machinery. Adopted from (6).

ment of the negative charged residues in the binding site. The statistic work done by our labo- ratory and others indicates that calcium has a preference to bind oxygen atoms provided by the carboxyl or hydroxyl groups from the sidechain or mainchain, which can be explained by its hard Lewis acid definition presenting less convalent interaction with a more rigid ionic radius. (43) Compared to other divalent metal ions such as zinc or cooper, the optimal coordination number of calcium is as high as seven.

Our lab has developed the grafting approach for probing site specific Ca2+ binding affinity. We have shown that CD2 is an excellent scaffold protein(42, 44-61). It retains the native struc- ture after the insertion of the EF-hand motif both in the absence and presence of Ca2+ ions. After optimizing the length of two glycine linkers that connect the Ca2+ binding loop and CD2 to provide sufficient freedom for the loop, we have shown that the grafted EF-hand loop retains its native Ca2+ binding property using high resolution NMR and 15N labeled protein(45). Contri- bution of flanking helices to the metal binding affinity of CaM have been investigated by in- serting the EF-loop, the loop with the existing F-helix, and the loop with both EF-helices of Site III of CaM into CD2. In contrast to the largely unfolded structure of the isolated peptide frag- ment, the inserted flanking helices are partially formed, as revealed by both CD and NMR. Ca2+ affinity is enhanced about 3-10 fold when the flanking helices are attached. We have first estimated the intrinsic Ca2+ _{affinities of the four EF-hand loops of CaM (I-IV) by individually}

grafting them into CD2. EF-loop I exhibits the strongest while EF-loop IV has the weakest bind- ing affinity for Ca2+, La3+, andTb3+. EF-loops I-IV of CaM has dissociation constants for Ca2+ of 34, 245, 185, and 814 µM, respectively. Based on the results, we proposed a charge-ligand- balanced model in which both the number of negatively charged ligand residues and the bal- anced electrostatic dentate-dentate repulsion by the adjacent charged residues are major de-

terminants for the Ca2+ _{binding affinities of EF-loops in CaM. Our grafting method provides a}

new strategy to obtain site-specific Ca2+ binding properties and to estimate the cooperativity and conformational change contributions of coupled EF-hand motifs. We have shown that the contribution of the cooperativity and conformational change to the Ca2+ affinity for the C- terminal is 40% greater than that for the N-terminal. The same approach will be used to probe the site-specific Ca2+ _{affinity and kinetic properties of engineered calcium binding proteins and}

sensors.

Based on the common features of calcium binding sites from detailed structural analy- sis of calcium binding proteins, our lab has successfully designed a calcium binding site on the surface of a beta-sheet non Ca2+ binding cell adhesion protein CD2 by site-directed mutage- nesis of four to five residues to negatively charged. The first generation of the designed Ca2+

binding CD2 variants having five negatively charged residues as the binding ligands, by fluo- rescent energy transfer between Tb3+ _{and aromatic residues Tyr81 and buried Trp32, the dis-}

sociation binding constant of the designed protein to Tb3+ was measured 21 µM, in comparison to 300 µM of a native protein gama-crystallin. Later, a more elegant work was completed by designing CD2 variants not rely on sequence or structural similarity between the recipient scaf- fold and a naturally evolved donor protein. Instead, the strategy takes into account the general local calcium-binding properties such as ligand types, charge, and the geometry of the primary coordination sphere and then identifies a constellation of backbone positions in the recipient scaffold that allows the introduction of the metal-binding site by mutations with potential ligand residues. The designed proteins were filtered using several criteria. First, the calcium should be solvent accessible. Second, the mutations should introduce little or no side-chain steric con- flicts with the preexisting atoms. Third, minimal disruption of hydrogen bonding and hydropho-

bic interactions is required. Fourth, three or four negatively charged residues at the primary coordination shell are preferred. The designed calcium-binding site is constructed by ligand residues from three different sequence regions (two anti-parallel beta-sheets and one from a flexible region) with a total of four negatively charged ligands. The metal selectivity of CD2.Ca1 was verified by metal replacement titration, and 1D NMR spectroscope. Adding 10 mM Ca2+ or 0.1 mM La3+ _{can significantly reduced the fluorescent intensity of CD2.Ca1 pre-loaded with 30}

µM Tb3+, while 10 mM Mg2+ or or 100 mM K+ reduced fluorescent intensity only 10-20%, in contrast to 5-10% fluorescent decrement by adding these metals without protein. The metal selectivity further verified by the 1D NMR spectra by observing conformational change induced by Ca2+ in the presence of other metal ions. And the Ca2+ /Mg2+ selectivity was verified by add- ing Ca2+ _{or Mg}2+ _{in the presence of 130 mM KCl. Mg}2+ _{does not induce conformational change}

even up to 10 mM, however, the spectra of 1.0 mM Ca2+ in the presence of 1.0 mM Mg2+ is identical to that of Ca2+ _{loaded form. A consequential work of designed Ca.CD2 was used to}

investigate the effect of designed Ca2+-binding sites on the biological function of CD2. There are different affinities of Ca.CD2-binding to CD48 by surface plasmon resonance in the ab- sence and presence of Ca2+_{. Using CD2 as the control (100%), the binding of Ca.CD2 to the}

conformation-dependent antibody OX34 is 104 and 106% with Ca2+ and EGTA, respectively. The binding to OX55 is 90 and 101% with Ca2+ _{and EGTA, respectively. These results strongly}

suggest that Ca.CD2 retains its native biological ability to bind to CD48. It is interesting to note that Ca2+ binding to Ca.CD2 decreases the binding affinity to CD48 by approximately 15%, al- though this designed site is located on the surface opposite from the CD48 recognition site. The binding of soluble CD48 results in significant changes in chemical shifts and line broaden- ing of a few residues at the C and C′ strands of domain 1 of CD2, as reported by Driscol and

colleagues. Although L63 is not located at the functional surface, upon binding to CD48, nuclei in this residue exhibit substantial chemical shift changes that are comparable to those of func- tional surface residues. The Ca2+ _{binding dissociation constant of Ca.CD2 was measured to}

1.4 mM by 2D NMR Ca2+ titration, while the Tb3+ and La3+ binding dissociation constant was 6.6 µM and 5.0 µM, respectively. Metal selectivity of Ca.CD2 between divalent and trivalent metal are more than 200 folds(47). The thermal stability of the designed CD2 variants was also investigated. The Tm of -5 negative charged residues of CD2 variants decreased to 41 °C in

compared to 61 °C of WT CD2, this decrement was partially restored by Ca2+ _{binding, without}

substantial conformational change of the secondary structure(62). The local conformational change of protein was investigated by Ca2+ and Ln3+ titration of CD2.6D79, with similar trend of Tm value changes in the presence and absence of Ca2+ _{(63), with tuned K}

ds ranging from tens

of micromolar to millimolar (47, 52). The secondary structure and spartial distribution of the de- signed Ca2+ _{binding ligands on the surface of CD2 are different from those in tranditional EF-}

hand based a 12-amino acid sequential loop which wraps around a Ca2+ ions, but protruding to the ourside of the protein with high solvent accessibility. We have further shown that calcium binding affinity can be changed by the ligand types and number of charged residues. Moreo- ver, the thermostability of the designed CD2 variants was significantly enhanced after Ca2+ binding investigated by the ITC of the decreased Tm values, in concondence with the computa-

tional simulation results (Yang, W. et. al., 2005, JACS).

In addition to calcium binding affinities and protein folding, metal selectivity is also im- portant. Mg2+ _{concentration in the intracellular environment is maintained at 0.3-0.6 mM(64)}

and the concentration of monovalent cations is in the 0.1-0.2 M range. In the resting eukaryotic cell, intracellular Mg2+ _{concentration is about10}3_{-fold higher than that of Ca}2+ ₍₁₀-3 _{M for Mg}2+

and 10-6 _{M for Ca}2+_{). Hence, calcium binding proteins such as EF-hand proteins are likely to be}

Mg2+ filled(65-67). Upon activation by the first messages, such as the binding of a hormone or a transmitter, or the passing of a Na+_/K+ _{nerve signal, a dramatic increase in calcium concen-}

tration in cells (from ~10-7 M at resting state to ~10-5 M at excited state) causes the removal of pre-bound Mg2+ of calmodulin and TnC upon calcium-binding. In contrast to the marked struc- tural transitions induced by Ca2+ _{binding, Mg}2+ _{binding causes only localized conformational}

changes within the four Ca2+-binding loops of CaM and not able to result in significant struc- tural effects required for the interaction of CaM with target proteins(67). The selectivity of pro- teins to Ca2+ over Mg2+ is defined as a ratio of their association constant, KCa/KMg. The values

of the ratio of KCa/KMg for the first two EF-hand motifs of CaM is about 2.5 x 102 (68). The values

of the KCa/KMg ratio for motif 3 and 4 of TnC and that of Ca-Mg sites of Parv are much higher,

around 4 x 103. The two motifs of the TnC (N-terminal) are Ca(II)-specific with KCa about 105 M- 1_{. Selectivity against these cations requires the calcium affinity to be superior by several or-}

ders of magnitude. In EF- proteins, Ca(II)/Mg(II) exchange appears closely related to physio- logical processes that involve cell excitation and relaxation, such as muscle contraction(69, 70).Therefore, In order to specifically detect calcium, biosensors are required to have strong metal selectivity. Currently, the metal selectivity of the genetically encoded calcium sensors has not been determined quantitatively, though a cytosolic Ca2+ _{sensor, TnC-XXL, created by}

replacing the Mg2+ sensitive domain of TnC by a nonsensitive domain, has been found to ex- hibit improved divalent metal selectivity.

In addition to calcium binding affinities and protein folding, metal selectivity is also impor- tant. The designed CD2 variants exhibited 102 metal selectivity between Ca2+ and Mg2+, sup- ported by the metal replacement titration between Tb3+ _{and Mg}2+ _{(citation). We will explore the}

Ca2+ _{binding affinities and metal selectivities by fluorescent intensity change and HSQC-NMR}

chemical shift perturbation with the established methods.The overall Ca2+ binding affinity of designed sensors will be obtained by fluorescence spectrophotometer (Photon Technology In- ternational, Inc.) with the purified protein samples, while the individual Kd of each ligand will be

investigated by chemical shift perturbation of HSQC spectra with NMR 600 MHz in our school as described in the preliminary results. The emitted fluorescent intensity as a function of Ca2+

concentration was fitted by equation 1 and 2 with 1:1 binding model(71), where F represents fraction of Ca2+_{-bound protein, R is the detected fluorescence intensity, [P]}

T is the total protein

concentration and Kd is the dissociate constant. In addition, metal selectivity will be investi- gated by purposely adding different metal ions such as Zn2+, Cu2+, Mg2+ with various concen- trations into protein solution, and examining whether designed sensor can differentiate these metals from calcium according to the fluorescent change. The structural basis of major classes of Mg2+ _{binding proteins, their capability in binding Ca}2+ _{and Mg}2+_/Ca2+ _{selectivity are reviewed}

in Chapter VI.