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Mycobacterium tuberculosis (M. tb) has a complex cell wall, composed largely of mycolic acids, that are crucial to its structural maintenance. The M. tb desaturase A1 (DesA1) is an essential Ca2+‐binding protein that catalyses a key step in mycolic acid biosynthesis. To investigate the structural and functional significance of Ca2+ binding, we introduced mutations at key residues in its Ca2+‐binding βγ‐crystallin motif to generate DesA1F303A, E304Q, and F303A‐E304Q. Complementation of a conditional ΔdesA1 strain of Mycobacterium smegmatis, with the Ca2+ non‐binders F303A or F303A‐E304Q, failed to rescue its growth phenotype; these complements also exhibited enhanced cell wall permeability. Our findings highlight the criticality of Ca2+ in DesA1 function, and its implicit role in the maintenance of mycobacterial cellular integrity. [ABSTRACT FROM AUTHOR]

Summary: We describe a set of proteins in which a βγ‐crystallin domain pairs with an Ig‐like domain, and which are confined to microbes, like bacteria, slime molds and fungi. DdCAD‐1 (Ca2+‐dependent cell adhesion molecule‐1) and abundant perithecial protein (APP) represent this class of molecules. Using the crystal structure of APP‐NTD (N‐terminal domain of APP), we describe its mode of Ca2+ binding and provide a generalized theme for correct identification of the Ca2+‐binding site within this class of molecules. As a common feature, one of the two Ca2+‐binding sites is non‐functional in the βγ‐crystallin domains of these proteins. While APP‐NTD binds Ca2+ with a micromolar affinity which is comparable to DdCAD‐1, APP surprisingly does not bind Ca2+. Crystal structures of APP and Ca2+‐bound APP‐NTD reveal that the interface interactions in APP render its Ca2+‐binding site inoperative. Thus, heterodomain association provides a novel mode of Ca2+‐binding regulation in APP. Breaking the interface interactions (mutating Asp30Ala, Leu132Ala and Ile135Ala) or separation from the Ig‐like domain removes the constraints upon the required conformational transition and enables the βγ‐crystallin domain to bind Ca2+. In mechanistic detail, our work demonstrates an interdomain interface adapted to distinct functional niches in APP and its homolog DdCAD‐1. A group of Ca2+‐binding proteins in which a βγ‐crystallin domain associates with Ig‐like domain, exists in selected bacteria, slime molds and fungi. We identify the mode of Ca2+ binding in these proteins. Despite the presence of a functional Ca2+‐binding motif, APP of Neurospora/Sorderia, a homolog of DdCAD‐1 of Dictyostelium, does not bind Ca2+. We also provide mechanistic details of the loss of Ca2+ binding in APP, where disability is rendered by interference from interdomain interface. [ABSTRACT FROM AUTHOR]

Ca 2+ regulation in living systems occurs via specific structural alterations, subtle or drastic, in the Ca 2+ -binding domains of sensor proteins. Sensor proteins perform designated nonredundant roles within the dense network of Ca 2+ -binding proteins. A detailed understanding of the structural changes in calcium sensor proteins due to Ca 2+ spikes that vary spatially, temporally, and in magnitude would provide better insights into the mechanism of Ca 2+ sensing. This chapter describes a method to study various stages during apo to the holo transition of Ca 2+ -binding proteins by Trp-mediated scanning of individual EF-hand motifs. We describe the applicability of this procedure to caldendrin, which is a neuronal Ca 2+ -binding protein and to integrin-binding protein. Tryptophan mutants of full-length caldendrin were designed to reveal local structural changes in each EF-hand of the protein. This method, referred to as "EF-hand scanning tryptophan mutagenesis," not only allows the identification of canonical and noncanonical EF-hands using very low concentrations of protein but also enables visualization of the hierarchical filling of Ca 2+ into the canonical EF-hands.

A crucial event in calcium signaling is the transition of a calcium sensor from the apo (Ca 2+ free) to the holo (Ca 2+ -saturated) state. Caldendrin (CDD) is a neuronal Ca 2+ -binding protein with two functional (EF3 and EF4) and two atypical (EF1 and EF2), non-Ca 2+ -binding EF-hand motifs. During the transition from the apo to the holo state, guided by the stepwise filling of Ca 2+ , the protein passes through distinct states and acquires a stable conformational state when only EF3 is occupied by Ca 2+ . This state is characterized by a Ca 2+ -derived structural gain in EF3 with destabilization of the EF4 motif. At higher Ca 2+ levels, when Ca 2+ fills in EF4, the motif regains stability. EF3 controls initial Ca 2+ binding and dictates structural destabilization of EF4. It is likely that this unexpected intermotif communication will have an impact on Ca 2+ -dependent target interactions.