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Leslie W-M Fung

Publications and source records attributed to Leslie W-M Fung.

5 recordsLinked to original sources
PubMed2005

Mutational effects at the tetramerization site of nonerythroid alpha spectrin.

Spectrin, a prominent cytoskeletal protein, exerts its fundamental role in cellular function by forming a sub-membrane filamentous network. An essential aspect of spectrin network formation is the tetramerization of spectrin alphabeta heterodimers. We used laboratory methods, the yeast two-hybrid system and random mutagenesis, to investigate, for the first time, effects of amino acid mutations on tetramerization of nonerythroid (brain) spectrin (fodrin). Based on high sequence homology with erythroid spectrin, we assume the putative tetramerization region of nonerythroid alpha-spectrin at the N-terminal region. We introduced mutations in the region consisting of residues 1-45 and studied mutational effects on spectrin alphabeta association to form tetramers. We detected single, double, and triple mutations involving 24 residues in this region. These amino acid mutations of nonerythroid alpha-spectrin exhibit full, partial, or no effect on the association with nonerythroid beta-spectrin. Single amino acid mutations in the region of residues 1-9 (D2Y, G5V, V6D, and V8M) did not affect the association. However, seven single mutations (I15F, I15N, R18G, V22D, R25P, Y26N, and R28P) affected the alphabeta association. These mutations were clustered in the region predicted by sequence alignment to be crucial in nonerythroid alpha-spectrin for tetramerization, a region that spanned residues 12-36, corresponding to the partial domain Helix C' (residues 21-45) in erythroid alpha-spectrin. In addition, two other mutations, one upstream and one downstream of this region at positions 10 (E10D) and 37 (R37P), also affected the alphabeta association. Our results implied nonerythroid alpha-spectrin partial domain helix may be longer than Helix C' (residues 21-45 and a total of 25 residues) in erythroid alpha-spectrin and spanned at least residues 10-37. It is interesting to note that seven out of these nine single mutations (I15F, I15N, R18G, V22D, R25P, Y26N, R37P) were at the a, d, e or g heptad positions based on sequence alignment with erythroid alpha-spectrin. Four of the mutated residues (I15, R18, V22, R25) are conserved in both erythroid and nonerythroid spectrin. These positions were previously identified as hot spots in erythroid alpha-spectrin that lead to severe hematological symptoms. This study clearly demonstrated that single mutation in a region predicted to be critical functionally in nonerythroid alpha-spectrin indeed leads to functional abnormalities and may lead to neurological disorders.

Amino Acid Sequence↗
PubMed2003

Structural analysis of the alpha N-terminal region of erythroid and nonerythroid spectrins by small-angle X-ray scattering.

We used SpalphaI-1-156 peptide, a well-characterized model peptide of the alphaN-terminal region of erythrocyte spectrin, and SpalphaII-1-149, an alphaII brain spectrin model peptide similar in sequence to SpalphaI-1-156, to study their association affinities with a betaI-spectrin peptide, SpbetaI-1898-2083, by isothermal titration calorimetry. We also determined their conformational flexibilities in solution by small-angle X-ray scattering (SAXS) methods. These two peptides exhibit sequence homology and could be expected to exhibit similar association affinities with beta-spectrin. However, our studies show that the affinity of SpalphaII-1-149 with SpbetaI-1898-2083 is much higher than that of SpalphaI-1-156. Our SAXS findings also indicate a significantly more extended conformation for SpalphaII-1-149 than for SpalphaI-1-156. The radius of gyration values obtained by two different analyses of SAXS data and by molecular modeling all show a value of about 25 A for SpalphaI-1-156 and of about 30 A for SpalphaII-1-149, despite the fact that SpalphaI-1-156 has seven amino acid residues more than SpalphaII-1-149. For SpalphaI-1-156, the SAXS results are consistent with a flexible junction between helix C' and the triple helical bundle that allows multiple orientations between these two structural elements, in good agreement with our published NMR analysis. The SAXS findings for SpalphaII-1-149 support the hypothesis that this junction region is rigid (and probably helical) for alphaII brain spectrin. The nature of the junction region, from one extreme as a random coil (conformationally mobile) segment in alphaI to another extreme as a rigid segment in alphaII, determines the orientation of helix C' relative to the first structural domain. We suggest that this particular junction region in alpha-spectrin plays a major role in modulating its association affinity with beta-spectrins, and thus regulates spectrin tetramer levels. We also note that these are the first conformational studies of brain spectrin.

Amino Acid Sequence↗
PubMed2003

Solution structural studies on human erythrocyte alpha-spectrin tetramerization site.

We have determined the solution NMR structure of a recombinant peptide that consists of the first 156 residues of erythroid alpha-spectrin. The first 20 residues preceding the first helix (helix C') are in a disordered conformation. The subsequent three helices (helices A1, B1, and C1) form a triple helical bundle structural domain that is similar, but not identical, to previously published structures for spectrin from Drosophila and chicken brain. Paramagnetic spin label-induced NMR resonance broadening shows that helix C', the partial domain involved in alpha- and beta-spectrin association, exhibits little interaction with the structural domain. Surprisingly, helix C' is connected to helix A1 of the structural domain by a segment of 7 residues (the junction region) that exhibits a flexible disordered conformation, in contrast to the predicted rigid helical structure. We suggest that the flexibility of this particular junction region may play an important role in modulating the association affinity of alpha- and beta-spectrin at the tetramerization site of different isoforms, such as erythroid spectrin and brain spectrin. These findings may provide insight for explaining various physiological and pathological conditions that are a consequence of varying alpha- and beta-subunit self-association affinities in their formation of the various spectrin tetramers.

Animals↗
PubMed2002

Nuclear magnetic resonance studies of mutations at the tetramerization region of human alpha spectrin.

Many spectrin mutations that destabilize tetramer formation and lead to hereditary hemolytic anemias are located at the N-terminal region of alpha-spectrin, with the Arg28 position considered to be a mutation hot spot. We have introduced mutations at positions 28 and 45 into a model peptide, Sp alpha 1-156, consisting of the first 156 residues in the N-terminal region of alpha-spectrin (alpha N). The association of these alpha-spectrin peptides that have single amino acid replacements with a beta-spectrin model peptide, consisting of the C-terminal region of beta-spectrin (beta C), was determined, and structural changes due to amino acid replacements were monitored by nuclear magnetic resonance (NMR). We found evidence for similar and very localized structural changes in Sp alpha 1-156Arg45Thr and Sp alpha 1-156Arg45Ser, although these 2 mutant peptides associated with beta-spectrin peptide with significantly differing affinities. The Sp alpha 1-156Arg28Ser peptide showed an affinity for the beta-spectrin peptide comparable to that of Sp alpha 1-156Arg45Ser, but it exhibited substantial and widespread spectral changes. Our results suggest that both Arg45 replacements induce only minor structural perturbations in the first helix of Sp alpha 1-156, but the Arg28Ser replacement affects both the first helix and the following structural domain. Our results also indicate that the mechanism for reduced spectrin tetramerization is through mutation-induced changes in molecular recognition at the alpha beta-tetramerization site, rather than through conformational disruption, as has been suggested in prior literature.

Amino Acid Substitution↗