Biomedical subjects

Hsiau-Wei Lee

Publications and source records attributed to Hsiau-Wei Lee.

9 recordsLinked to original sources

PubMed2006

Prediction of EF-hand calcium-binding proteins and analysis of bacterial EF-hand proteins.

The EF-hand protein with a helix-loop-helix Ca(2+) binding motif constitutes one of the largest protein families and is involved in numerous biological processes. To facilitate the understanding of the role of Ca(2+) 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 Ca(2+)-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 Ca(2+) 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 Ca(2+) binding motifs provides not only an insight into the role of Ca(2+) and Ca(2+)-binding proteins in bacterial systems, but also a way to explore and define the role of Ca(2+) in other biological systems (calciomics).

Amino Acid Motifs↗

PubMed2006

Functional elements on SIRPalpha IgV domain mediate cell surface binding to CD47.

SIRPalpha and SIRPbeta1, the two major isoforms of the signal regulatory protein (SIRP) family, are co-expressed in human leukocytes but mediate distinct extracellular binding interactions and divergent cell signaling responses. Previous studies have demonstrated that binding of SIRPalpha with CD47, another important cell surface molecule, through the extracellular IgV domain regulates important leukocyte functions including macrophage recognition, leukocyte adhesion and transmigration. Although SIRPbeta1 shares highly homologous extracellular IgV structure with SIRPalpha, it does not bind to CD47. Here, we defined key amino acid residues exclusively expressing in the IgV domain of SIRPalpha, but not SIRPbeta1, which determine the extracellular binding interaction of SIRPalpha to CD47. These key residues include Gln67, a small hydrophobic amino acid (Ala or Val) at the 57th position and Met102. We found that Gln67 and Ala/Val57 are critical. Mutation of either of these residues abates SIRPalpha directly binding to CD47. Functional cell adhesion and leukocyte transmigration assays further demonstrated central roles of Gln67 and Ala/Val57 in SIRPalpha extracellular binding mediated cell interactions and cell migration. Another SIRPalpha-specific residue, Met102, appears to assist SIRPalpha IgV binding through Gln67 and Ala/Val57. An essential role of these amino acid residues in SIRPalpha binding to CD47 was further confirmed by introducing these residues into the SIRPbeta1 IgV domain, which dramatically converts SIRPbeta1 into a CD47-binding molecule. Our results thus revealed the molecular basis by which SIRPalpha binds to CD47 and shed new light into the structural mechanisms of SIRP isoform mediated distinctive extracellular interactions and cellular responses.

Amino Acid Sequence↗

PubMed2005

Probing site-specific calmodulin calcium and lanthanide affinity by grafting.

Ca2+ binding is essential for the biological functions of calmodulin (CaM) as a trigger/sensor protein to regulate many biological processes in the Ca2+ -signaling cascade. A challenge in understanding the mechanism of Ca2+ signaling is to obtain site-specific information about the Ca2+ binding properties of individual Ca2+ -binding sites of EF-hand proteins, especially for CaM. In this paper, we report the first estimation of the intrinsic Ca2+ affinities of the four EF-hand loops of calmoduin (I-IV) by individually grafting into the domain 1 of CD2. Taking advantage of the Trp residues in the host protein, we first determined metal-binding affinities for Tb3+, Ca2+, and La3+ for all four grafted EF-loops using Tb3+ aromatic resonance energy transfer. EF-loop I exhibits the strongest binding affinity for Ca2+, La3+, and Tb3+, while EF-loop IV has the weakest metal-binding affinity. EF-loops I-IV of CaM have dissociation constants for Ca2+ of 34, 245, 185, and 814 microM, respectively, with the order I > III approximately equal to II > IV. These findings support a charge-ligand-balanced model in which both the number of negatively charged ligand residues and the balanced electrostatic dentate-dentate repulsion by the adjacent charged residues are two major determinants for the relative Ca2+ -binding affinities of EF-loops in CaM. Our grafting method provides a new strategy to obtain site-specific Ca2+ binding properties and a better estimation of the cooperativity and conformational change contributions of coupled EF-hand proteins.

Animals↗

PubMed2005

Calcium and lanthanide affinity of the EF-loops from the C-terminal domain of calmodulin.

To obtain site-specific information about individual EF-hand motifs, the EF-hand Ca(2+)-binding loops from site III and site IV of calmodulin (CaM) were inserted separately into a non-Ca(2+)-binding cell adhesion protein, domain 1 of CD2 (denoted as CaM-CD2-III-5G-52 and CaM-CD2-IV-5G-52). Structural analyses using various spectroscopic methods have shown that the host protein CD2 retains its native structure after the insertion of the 12-residue loops. The Tb(3+) fluorescence enhancement upon formation of a Tb(3+)-protein complex and the direct competition by La(3+) and Ca(2+) suggest that native Ca(2+)-binding pockets are formed in both engineered proteins. Moreover, as revealed by NMR, both Ca(2+) and La(3+) specifically interact with the residues at the grafted EF-loop. The CaM-CD2-III-5G-52 has stronger affinities to Ca(2+), Tb(3+) and La(3+) than CaM-CD2-IV-5G-52, indicating differential intrinsic metal-binding affinities of the EF-loops.

Amino Acid Sequence↗

PubMed2003

Rational design of a calcium-binding protein.

Calcium ions play key roles as structural components in biomineralization and as a second messenger in signaling pathways. We have introduced a de novo designed calcium-binding site into the framework of a non-calcium-binding protein, domain 1 of CD2. The resulting protein selectively binds calcium over magnesium with calcium-binding affinity comparable to that of natural extracellular calcium-binding proteins (K(d) of 50 microM). This experiment is the first successful metalloprotein design that has a high coordination number (seven) metal-binding site constructed into a beta-sheet protein. Our results demonstrate the feasibility of designing a single calcium-binding site into a host protein, taking into account only local properties of a calcium-binding site obtained by a survey of natural calcium-binding proteins and chelators. The resulting site exhibits strong metal selectivity, suggesting that it should now be feasible to understand and manipulate signaling processes by designing novel calcium-modulated proteins with specifically desired functions and to affect their stability.

Binding Sites↗

PubMed2003

A grafting approach to obtain site-specific metal-binding properties of EF-hand proteins.

The EF-hand calcium-binding loop III from calmodulin was inserted with glycine linkers into the scaffold protein CD2.D1 at three locations to study site-specific calcium binding properties of EF-hand motifs. After insertion, the host protein retains its native structure and forms a 1:1 metal-protein complex for calcium and its analog, lanthanum. Tyrosine-sensitized Tb3+ energy transfer exhibits metal binding and La3+ and Ca2+ compete for the metal binding site. The grafted EF-loop III in different environments has similar La3+ binding affinities, suggesting that it is largely solvated and functions independently from the host protein.

Animals↗

PubMed2002

Isolated EF-loop III of calmodulin in a scaffold protein remains unpaired in solution using pulsed-field-gradient NMR spectroscopy.

Calmodulin (CaM) is a trigger calcium-dependent protein that regulates many biological processes. We have successfully engineered a series of model proteins, each containing a single EF-hand loop but with increasing numbers of Gly residues linking the EF-hand loop to a scaffold protein, cluster of differentiation 2 (CD2), to obtain the site-specific calcium-binding ability of a protein with EF-hand motifs without the interference of cooperativity. Loop III of calmodulin with two Gly linkers in CD2 (CaM-CD2-III-5G) has metal affinities with K(d) values of 1.86 x 10(-4) and 5.8 x 10(-5) M for calcium and lanthanum, respectively. The oligomeric states of the CD2 variants were examined by pulsed-field-gradient nuclear magnetic resonance (PFG NMR). The diffusion coefficient values of CD2 variants are about 11.1 x 10(-7) cm(2)/s both in the presence and absence of metal ions, which are the same as that of wild-type CD2. This suggests that the isolated EF-loop III of calmodulin inserted in the scaffold protein is able to bind calcium and lanthanum as a monomer, which is in contrast to the previous observation of the EF-hand motif. Our results imply that additional factors that reside outside of the EF-loop III may contribute to the pairing of EF-hand motifs of calmodulin. This result is of interest as it opens up the way for studying the ion-binding properties of isolated EF-hands, which in turn can answer important questions about the properties of EF-hands, the large and important group of calcium-binding signaling proteins.

Binding Sites↗

PubMed2002

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 calbindin(D9K) 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.

Algorithms↗

PubMed2002

Metal-binding studies for a de novo designed calcium-binding protein.

To understand the key determinants in calcium-binding affinity, a calcium-binding site with pentagonal bipyramid geometry was designed into a non-calcium-binding protein, domain 1 of CD2. This metal-binding protein has five mutations with a net charge in the coordination sphere of -5 and is termed DEEEE. Fluorescence resonance energy transfer was used to determine the metal-binding affinity of DEEEE to the calcium analog terbium. The addition of protein concentration to Tb(III) solution results in a large enhancement of Tb(III) fluorescence due to energy transfer between terbium ions and aromatic residues in CD2-D1. In addition, both calcium and lanthanum compete with terbium for the same desired metal binding pocket. Our designed protein exhibits a stronger affinity for Tb(III), with a K(d) of 21 microM, than natural calcium-binding proteins with a similar Greek key scaffold.

Amino Acids↗