The steroidogenic acute regulatory protein-related lipid transfer (START) domain 
family is defined by a conserved 210-amino acid sequence that folds into an α/β 
helix-grip structure. Members of this protein family bind a variety of ligands, 
including cholesterol, phospholipids, sphingolipids, and bile acids, with 
putative roles in nonvesicular lipid transport, metabolism, and cell signaling. 
Among the soluble START proteins, STARD4 is expressed in most tissues and has 
previously been shown to transfer sterol, but the molecular mechanisms of 
membrane interaction and sterol binding remain unclear. In this work, we use 
biochemical techniques to characterize regions of STARD4 and determine their 
role in membrane interaction and sterol binding. Our results show that STARD4 
interacts with anionic membranes through a surface-exposed basic patch and that 
introducing a mutation (L124D) into the Omega-1 (Ω1) loop, which covers the 
sterol binding pocket, attenuates sterol transfer activity. To gain insight into 
the attenuating mechanism of the L124D mutation, we conducted structural and 
biophysical studies of wild-type and L124D STARD4. These studies show that the 
L124D mutation reduces the conformational flexibility of the protein, resulting 
in a diminished level of membrane interaction and sterol transfer. These studies 
also reveal that the C-terminal α-helix, and not the Ω1 loop, partitions into 
the membrane bilayer. On the basis of these observations, we propose a model of 
STARD4 membrane interaction and sterol binding and release that requires dynamic 
movement of both the Ω1 loop and membrane insertion of the C-terminal α-helix.