How Ribosomes Team Up With NAC to Tag Proteins for Membrane Docking

N-glycine myristoylation—the attachment of a 14-carbon fatty acid to the N-terminus of newly synthesized proteins—is critical for enabling proteins to reversibly dock with cellular membranes and participate in signaling, stress responses, and immune function. This modification is carried out co-translationally by N-myristoyltransferases (NMT1 and NMT2), enzymes that must act while the protein is still being assembled on the ribosome. Dysregulation of NMT2 specifically has been linked to cardiac hypertrophy and heart failure, with patient cardiomyocytes showing up to 60% reductions in NMT2 levels, as well as certain cancers. Despite its biomedical importance, the molecular mechanism by which NMT2 is recruited to ribosomes and gains access to nascent substrates had remained unknown.

To address this gap, Zdancewicz and colleagues first confirmed using sucrose gradient centrifugation in HEK293 cells that NMT2 co-localizes with ribosomal fractions in living human cells. They then performed in vitro binding assays with purified human ribosomes and showed NMT2 binds ribosomes directly, with binding approximately doubled in the presence of the heterodimeric nascent polypeptide-associated complex (NAC). Using ribosome-nascent chain complexes (RNCs) loaded with varying lengths of MARCKS—an NMT2-specific substrate—the team found that NMT2 binding was relatively consistent across nascent chain lengths, with a modest peak at 74 amino acids, the length chosen for structural studies.

The centerpiece of the study is a high-resolution cryo-EM structure of the ternary RNC:NMT2:NAC complex. The structure reveals that NMT2 and NAC together form an extended channel directly continuous with the ribosomal polypeptide exit tunnel, physically guiding the nascent chain from the tunnel into NMT2's catalytic pocket. Strikingly, the cryo-EM density shows the first nine amino acids of the MARCKS substrate seated in the catalytic site, with visible side chains and electrostatic interactions between substrate residues and NMT2. Coenzyme A density is also resolved in the active site, capturing an intermediate state of the reaction.

The structure also uncovers a previously unappreciated ribosomal RNA clamp: helix 59 of the 28S rRNA wraps around NMT2 to anchor it on the ribosomal surface, orienting its catalytic site toward the tunnel exit. The C-terminal tail of NACβ makes direct contacts with NMT2, and truncation experiments confirmed this tail is required for efficient NMT2 recruitment. Similarly, the N-terminal tail of NMT2 contributes to ribosome binding, and its deletion reduces association. Extensive contacts between NMT2, NAC, and multiple ribosomal proteins (including uL22, uL24, and eL39) stabilize the complex further.

These findings establish NAC as a master coordinator that sequentially recruits methionine aminopeptidase (which cleaves the initiator methionine to expose glycine) and then NMT2 to the ribosome, ensuring ordered co-translational modification. The mechanistic blueprint provided here opens avenues for structure-guided drug design targeting NMT2-ribosome interactions in heart disease and cancer, and raises questions about how NMT1 and NMT2 compete or cooperate for substrates in different tissues.