Staphylococcus aureus (S. aureus) is a major Gram-positive pathogen, and treatment of S. aureus infections is often challenging due to widespread antibiotic resistance. In Gram-positive bacteria such as S. aureus, D-alanylation of teichoic acids (TA) reduces the net negative charge of the cell envelope and contributes to resistance to diverse antibiotics, particularly cationic antimicrobial peptides. D-alanylation is mediated by the dltABCD operon, which encodes four proteins (DltA, DltB, DltC, and DltD), all of which is essential for the multistep transfer of D-alanine to teichoic acids. Here, we present the first crystal structure of the S. aureus D-alanyl carrier protein DltC and analyze its interaction with DltA using AlphaFold3 and all-atom molecular dynamics simulations. We further show that single substitutions of SaDltA-SaDltC interface residues abolish SaDltC mediated enhancement of SaDltA catalysis. Together, these findings define a catalytically critical S. aureus DltA-DltC interface and provide a structural insight for targeting the D-alanylation pathway as a potential anti-Staphylococcus strategy.

In the periplasmic space of Gram-negative bacteria, located between the inner cytoplasmic membrane and the bacterial outer membrane, a variety of proteins are present, and their regulation is critical for bacterial survival [ 1]. Among these, the HtrA family of proteins function both as proteases and chaperones, playing important roles in maintaining bacterial homeostasis and pathogenicity, particularly under stressful conditions such as nutritional deprivation and elevated temperatures. To exert those functions, HtrA family proteins have been shown to form polyhedral cage structures (e.g., tetrahedral or octahedral), when they encounter diverse substrates, enabling them to perform either protease or chaperone functions [ 2]. Previously, we obtained high-resolution cryogenic electron microscopy (cryo-EM) structures of HtrA proteins, which revealed the soft-hinge rigid-body mechanism that underlies the self-assembly of HtrA proteins into polyhedral cages [ 3]. To achieve highly monodisperse cage particles, we utilized soft, less rigid client proteins that can modulate the inherent chaperone activity of HtrA proteins, enabling the efficient efficient formation of polymorphic cages, tailored to the size of the client proteins. By reconstructing the atomic-resolution cryo-EM structures using obtained tetrahedral and octahedral clusters, the polymorphic clustering of HtrA family enzymes is validated in terms of soft and rigid domains, revealing effective routes for protein self-assemblies with procedural flexibility. Here, we further engineered a HtrA protein for selective targeting and delivery of diverse materials specifically to specific target cells. By either functionalizing the outer surface of the cage and modifying the internal cavity, we demonstrate the ability to target specific receptor-overexpressing cell types, which may facilitate targeted drug delivery [4].