Human steroid 5α-reductase 2 (SRD5A2) is an integral membrane enzyme in steroid metabolism and catalyzes the reduction of testosterone to dihydrotestosterone. Mutations in the SRD5A2 gene have been linked to 5α-reductase deficiency and prostate cancer. Finasteride and dutasteride, as SRD5A2 inhibitors, are widely used antiandrogen drugs for benign prostate hyperplasia. The molecular mechanisms underlying enzyme catalysis and inhibition for SRD5A2 and other eukaryotic integral membrane steroid reductases remain elusive due to a lack of structural information. Here, we report a crystal structure of human SRD5A2 at 2.8 Å, revealing a unique 7-TM structural topology and an intermediate adduct of finasteride and NADPH as NADP-dihydrofinasteride in a largely enclosed binding cavity inside the transmembrane domain. Structural analysis together with computational and mutagenesis studies reveal the molecular mechanisms of the catalyzed reaction and of finasteride inhibition involving residues E57 and Y91. Molecular dynamics simulation results indicate high conformational dynamics of the cytosolic region that regulate NADPH/NADP+ exchange. Mapping disease-causing mutations of SRD5A2 to our structure suggests molecular mechanisms for their pathological effects. Our results offer critical structural insights into the function of integral membrane steroid reductases and may facilitate drug development.

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The signaling of prostaglandin D (PGD) through G-protein-coupled receptor (GPCR) CRTH2 is a major pathway in type 2 inflammation. Compelling evidence suggests the therapeutic benefits of blocking CRTH2 signaling in many inflammatory disorders. Currently, a number of CRTH2 antagonists are under clinical investigation, and one compound, fevipiprant, has advanced to phase 3 clinical trials for asthma. Here, we present the crystal structures of human CRTH2 with two antagonists, fevipiprant and CAY10471. The structures, together with docking and ligand-binding data, reveal a semi-occluded pocket covered by a well-structured amino terminus and different binding modes of chemically diverse CRTH2 antagonists. Structural analysis suggests a ligand entry port and a binding process that is facilitated by opposite charge attraction for PGD, which differs significantly from the binding pose and binding environment of lysophospholipids and endocannabinoids, revealing a new mechanism for lipid recognition by GPCRs.

Kidney ankyrin repeat-containing proteins (KANK1/2/3/4) belong to a family of scaffold proteins, playing critical roles in cytoskeleton organization, cell polarity, and migration. Mutations in KANK proteins are implicated in cancers and genetic diseases, such as nephrotic syndrome. KANK proteins can bind various target proteins through different protein regions, including a highly conserved ankyrin repeat domain (ANKRD). However, the molecular basis for target recognition by the ANKRD remains elusive. In this study, we solved a high-resolution crystal structure of the ANKRD of KANK1 in complex with a short sequence of the motor protein kinesin family member 21A (KIF21A), revealing that the highly specific target-binding mode of the ANKRD involves combinatorial use of two interfaces. Mutations in either interface disrupted the KANK1-KIF21A interaction. Cellular immunofluorescence localization analysis indicated that binding-deficient mutations block recruitment of KIF21A to focal adhesions by KANK1. In conclusion, our structural study provides mechanistic explanations for the ANKRD-mediated recognition of KIF21A and for many disease-related mutations identified in human KANK proteins.