Jenny J. Yang

Structural analysis, identification, and design of calcium-binding sites in proteins

Assigning proteins with functions based on the 3‐D structure requires high‐speed techniques to make a systematic survey of protein structures. Calcium regulates many biological systems by binding numerous proteins in different biological environments. Despite the great diversity in the composition of ligand residues and bond angles and lengths of calcium‐binding sites, our structural analysis of 11 calcium‐binding sites in different classes of proteins has shown that common local structural parameters can be used to identify and design calcium‐binding proteins. Natural calcium‐binding sites in both EF‐hand proteins and non‐EF‐hand proteins can be described with the smallest deviation from the geometry of an ideal pentagonal bipyramid. Further, two different magnesium‐binding sites in parvalbumin and calbindinD9K can also be identified using an octahedral geometry. Using the established method, we have designed de novo calcium‐binding sites into the scaffold of non‐calcium‐binding proteins CD2 and Rop. Our results suggest that it is possible to identify calcium‐ and magnesium‐binding sites in proteins and design de novo metal‐binding sites. Proteins 2002;47:344–356.

Prediction of EF‐hand calcium‐binding proteins and analysis of bacterial EF‐hand proteins

The EF‐hand protein with a helix–loop–helix Ca2+ binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca2+ in biological systems using genomic information, we report, herein, our improvement on the pattern search method for the identification of EF‐hand and EF‐like Ca2+‐binding proteins. The canonical EF‐hand patterns are modified to cater to different flanking structural elements. In addition, on the basis of the conserved sequence of both the N‐ and C‐terminal EF‐hands within S100 and S100‐like proteins, a new signature profile has been established to allow for the identification of pseudo EF‐hand and S100 proteins from genomic information. The new patterns have a positive predictive value of 99% and a sensitivity of 96% for pseudo EF‐hands. Furthermore, using the developed patterns, we have identified zero pseudo EF‐hand motif and 467 canonical EF‐hand Ca2+ binding motifs with diverse cellular functions in the bacteria genome. The prediction results imply that pseudo EF‐hand motifs are phylogenetically younger than canonical EF‐hand motifs. Our prediction of Ca2+ binding motifs provides not only an insight into the role of Ca2+ and Ca2+‐binding proteins in bacterial systems, but also a way to explore and define the role of Ca2+ in other biological systems (calciomics). Proteins 2006.

Multiple Ca(2+)-binding sites in the extracellular domain of the Ca(2+)-sensing receptor corresponding to cooperative Ca(2+) response.

A small change in the extracellular Ca(2+) concentration ([Ca(2+)](o)) integrates cell signaling responses in multiple cellular and tissue networks and functions via activation of Ca(2+)-sensing receptors (CaSR). Mainly through binding of Ca(2+) to the large extracellular domain (ECD) of the dimeric CaSR, intracellular Ca(2+) responses are highly cooperative with an apparent Hill coefficient ranging from 2 to 4. We have previously reported the identification of two continuous putative Ca(2+)-binding sites by grafting CaSR-derived, Ca(2+)-binding peptides to a scaffold protein, CD2, that does not bind Ca(2+). In this paper, we predict more potential noncontinuous Ca(2+)-binding sites in the ECD. We dissect the intact CaSR into three globular subdomains, each of which contains two to three predicted Ca(2+)-binding sites. This approach enables us to further understand the mechanisms underlying the binding of multiple metal ions to extended polypeptides derived from a location within the ECD of the CaSR, which would be anticipated to more closely mimic the structure of the native CaSR ECD. Tb(3+) luminescence energy transfer, ANS fluorescence, and NMR studies show biphasic metal-binding components and Ca(2+)-dependent conformational changes in these subdomains. Removing the predicted Ca(2+)-binding ligands in site 1 and site 3 abolishes the first binding step and second binding step, respectively. Studies on these subdomains suggest the existence of multiple metal-binding sites and metal-induced conformational changes that might be responsible for the switching on and off the CaSR by the transition between its open inactive form and closed active form.

Viral calciomics: interplays between Ca2+ and virus.

Abstract Ca2+ is one of the most universal and versatile signaling molecules and is involved in almost every aspect of cellular processes. Viruses are adept at utilizing the universal Ca2+ signal to create a tailored cellular environment that meets their own demands. This review summarizes most of the known mechanisms by which viruses perturb Ca2+ homeostasis and utilize Ca2+ and cellular Ca2+-binding proteins to their benefit in their replication cycles. Ca2+ plays important roles in virion structure formation, virus entry, viral gene expression, posttranslational processing of viral proteins and virion maturation and release. As part of the review, we introduce an algorithm to identify linear “EF-hand” Ca2+-binding motifs which resulted in the prediction of a total of 93 previously unrecognized Ca2+-binding motifs in virus proteins. Many of these proteins are nonstructural proteins, a class of proteins among which Ca2+ interactions had not been formerly appreciated. The presence of linear Ca2+-binding motifs in viral proteins enlarges the spectrum of Ca2+–virus interplay and expands the total scenario of viral calciomics.

Identification of 70 calcium-sensing receptor mutations in hyper- and hypo-calcaemic patients: evidence for clustering of extracellular domain mutations at calcium-binding sites

The calcium-sensing receptor (CaSR) is a G-protein-coupled receptor that has an extracellular bilobed venus flytrap domain (VFTD) predicted to contain five calcium (Ca(2+))-binding sites. To elucidate the structure-function relationships of the VFTD, we investigated 294 unrelated probands with familial hypocalciuric hypercalcaemia (FHH), neonatal severe primary hyperparathyroidism (NSHPT) or autosomal dominant hypocalcaemic hypercalciuria (ADHH) for CaSR mutations and performed in vitro functional expression studies and three-dimensional modelling of mutations involving the VFTD. A total of 70 different CaSR mutations were identified: 35 in FHH, 10 in NSHPT and 25 in ADHH patients. Furthermore, a CaSR variant (Glu250Lys) was identified in FHH and ADHH probands and demonstrated to represent a functionally neutral polymorphism. NSHPT was associated with a large proportion of truncating CaSR mutations that occurred in the homozygous or compound heterozygous state. Thirty-four VFTD missense mutations were identified, and 18 mutations were located within 10 Å of one or more of the predicted Ca(2+)-binding sites, particularly at the VFTD cleft, which is the principal site of Ca(2+) binding. Mutations of residues 173 and 221, which are located at the entrance to the VFTD cleft binding site, were associated with both receptor activation (Leu173Phe and Pro221Leu) and inactivation (Leu173Pro and Pro221Gln), thereby highlighting the importance of these residues for entry and binding of Ca(2+) by the CaSR. Thus, these studies of disease-associated CaSR mutations have further elucidated the role of the VFTD cleft region in Ca(2+) binding and the function of the CaSR.

Identification and Dissection of Ca2+-binding Sites in the Extracellular Domain of Ca2+-sensing Receptor

Ca2+-sensing receptors (CaSRs) represent a class of receptors that respond to changes in the extracellular Ca2+ concentration ([Ca2+]o) and activate multiple signaling pathways. A major barrier to advancing our understanding of the role of Ca2+ in regulating CaSRs is the lack of adequate information about their Ca2+-binding locations, which is largely hindered by the lack of a solved three-dimensional structure and rapid off rates due to low Ca2+-binding affinities. In this paper, we have reported the identification of three potential Ca2+-binding sites in a modeled CaSR structure using computational algorithms based on the geometric description and surface electrostatic potentials. Mutation of the predicted ligand residues in the full-length CaSR caused abnormal responses to [Ca2+]o, similar to those observed with naturally occurring activating or inactivating mutations of the CaR, supporting the essential role of these predicted Ca2+-binding sites in the sensing capability of the CaSR. In addition, to probe the intrinsic Ca2+-binding properties of the predicted sequences, we engineered two predicted continuous Ca2+-binding sequences individually into a scaffold protein provided by a non-Ca2+-binding protein, CD2. We report herein the estimation of the metal-binding affinities of these predicted sites in the CaSR by monitoring aromatic-sensitized Tb3+ fluorescence energy transfer. Removing the predicted Ca2+-binding ligands resulted in the loss of or significantly weakened cation binding. The potential Ca2+-binding residues were shown to be involved in Ca2+/Ln3+ binding by high resolution NMR and site-directed mutagenesis, further validating our prediction of Ca2+-binding sites within the extracellular domain of the CaSR.

Rational Design of Protein-based MRI Contrast Agents

We describe the rational design of a novel class of magnetic resonance imaging (MRI) contrast agents with engineered proteins (CAi.CD2, i = 1, 2,..., 9) chelated with gadolinium. The design of protein-based contrast agents involves creating high-coordination Gd(3+) binding sites in a stable host protein using amino acid residues and water molecules as metal coordinating ligands. Designed proteins show strong selectivity for Gd(3+) over physiological metal ions such as Ca(2+), Zn(2+), and Mg(2+). These agents exhibit a 20-fold increase in longitudinal and transverse relaxation rate values over the conventional small-molecule contrast agents, e.g., Gd-DTPA (diethylene triamine pentaacetic acid), used clinically. Furthermore, they exhibit much stronger contrast enhancement and much longer blood retention time than Gd-DTPA in mice. With good biocompatibility and potential functionalities, these protein contrast agents may be used as molecular imaging probes to target disease markers, thereby extending applications of MRI.

Identification of the Calmodulin Binding Domain of Connexin 43

Calmodulin (CaM) has been implicated in mediating the Ca2+-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136–158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca2+-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its α-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K+ is in the range of 0.7–1 μm. Upon binding of the peptide to CaM, the apparent Kd of Ca2+ for CaM decreased from 2.9 ± 0.1 to 1.6 ± 0.1 μm, and the Hill coefficient nH increased from 2.1 ± 0.1 to 3.3 ± 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca2+-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136–158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca2+-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca2+-dependent manner, providing a molecular basis for the well characterized Ca2+-dependent inhibition of Cx43-containing gap junctions.

Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments

Quantitative analysis of Ca2+ fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca2+-dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca2+ binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca2+ release kinetics with an off-rate estimated at around 700 s−1 and appropriate Ca2+ binding affinity, likely attributable to local Ca2+-induced conformational changes around the designed Ca2+ binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca2+ dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca2+ in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca2+ release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca2+ homeostasis.

Predicting calcium-binding sites in proteins—A graph theory and geometry approach

Identifying calcium‐binding sites in proteins is one of the first steps towards predicting and understanding the role of calcium in biological systems for protein structure and function studies. Due to the complexity and irregularity of calcium‐binding sites, a fast and accurate method for predicting and identifying calcium‐binding protein is needed. Here we report our development of a new fast algorithm (GG) to detect calcium‐binding sites. The GG algorithm uses a graph theory algorithm to find oxygen clusters of the protein and a geometric algorithm to identify the center of these clusters. A cluster of four or more oxygen atoms has a high potential for calcium binding. High performance with about 90% site sensitivity and 80% site selectivity has been obtained for three datasets containing a total of 123 proteins. The results suggest that a sphere of a certain size with four or more oxygen atoms on the surface and without other atoms inside is necessary and sufficient for quickly identifying the majority of the calcium‐binding sites with high accuracy. Our finding opens a new avenue to visualize and analyze calcium‐binding sites in proteins facilitating the prediction of functions from structural genomic information. Proteins 2006.