NMT2 Human

What is the structural composition of human NMT2?

The crystal structure of human NMT2 has been solved at high resolution (PDB: 4C2X), providing detailed insights into its structural organization . The enzyme contains a conserved substrate-binding pocket that accommodates both peptide substrates and myristoyl-CoA. A co-crystal structure of NMT2 with a KVLSKIF peptide and myristoyl-CoA at 1.93 Å resolution demonstrates the mechanism of lysine myristoylation, clearly showing that the lysine ε-amine (not α-amine) is the site of modification . The simulated-annealing omit map unambiguously demonstrates the covalent bond between the lysine and myristoyl group, with concomitant loss of electron density connecting the myristoyl group and CoA .

Comparison with previous NMT structures reveals that lysine binds analogously to glycine substrates, with the amide bond directly overlapping between the structures . Interestingly, the adenosine-3′-phosphate region of CoA bound to NMT2 shows minimal electron density, suggesting flexibility or hydrolysis at the 5′ phosphate . Similarly, the region from Arg115 to His135, involved in adenosine binding, also exhibits flexibility and was not modeled in some structures .

What is the primary function of human NMT2?

NMT2 catalyzes protein N-myristoylation, a co- and post-translational modification that plays critical roles in protein localization, stability, and function. The primary functions include:

• Catalyzing the attachment of myristoyl groups to N-terminal glycine residues of proteins following methionine removal

• Myristoylating specific lysine residues, such as K3 in ARF6

• Regulating membrane association of substrate proteins

• Participating in diverse cellular processes including vesicular trafficking and signal transduction

The recently discovered ability of NMT2 to modify internal lysine residues significantly expands its functional repertoire and regulatory potential in cellular processes . For example, NMT2-mediated lysine myristoylation of ARF6 on K3 allows it to remain on membranes during the GTPase cycle, explaining the puzzling dissimilarity of ARF6 to other ARF proteins in terms of membrane association .

What are the known substrates of human NMT2?

NMT2 has a diverse range of substrates, including:

• Well-characterized substrates:

  • ARF6 (ADP-ribosylation factor 6) is myristoylated at both G2 and K3

  • Other ARF proteins (ARF1-5) are myristoylated at their N-terminal glycine residues

• Global profiling studies:

  • Quantitative chemical proteomics combined with NMT inhibition has identified >100 N-myristoylated proteins in human cells, >95% of which were identified for the first time at endogenous levels

• Potential lysine myristoylation substrates:

  • NMT2 may regulate other substrates with lysine at position 3 or 4

  • Insertion experiments show that adding one glycine between A2 and K3 in ARF6 G2A is tolerated, while insertion of alanine significantly decreases myristoylation

The discovery that NMT2 can myristoylate specific lysine residues significantly expands the potential substrate pool beyond the traditional N-terminal glycine-containing proteins.

How is NMT2 activity regulated in cells?

NMT2 activity is regulated through multiple mechanisms:

• Substrate-dependent regulation:

  • NMT2 shows preference for GTP-bound forms of substrates like ARF6

  • Co-immunoprecipitation experiments demonstrate that NMT2 preferentially binds to the active ARF6 Q67L mutant (GTP-bound form) over the inactive T27N mutant (GDP-bound form)

  • Co-localization studies confirm that NMT2 associates better with ARF6 Q67L than with ARF6 T27N

• Coordinated action with deacylases:

  • SIRT2 (a NAD+-dependent deacylase) removes myristoyl groups from lysine residues

  • This creates a dynamic cycle of myristoylation-demyristoylation that is coupled to the GTPase cycle of substrates like ARF6

This selective preference of NMT2 for active (GTP-bound) ARF6 and SIRT2 for inactive (GDP-bound) ARF6 creates a sophisticated regulatory system that coordinates lysine myristoylation-demyristoylation with the GTPase cycle.

How do researchers distinguish between NMT1 and NMT2 activity in experimental settings?

Distinguishing between NMT1 and NMT2 activities requires sophisticated experimental approaches:

• Genetic manipulation approaches:

  • Selective knockdown of NMT1 or NMT2 using siRNA or shRNA followed by rescue experiments

  • In studies with ARF6 G2A (which can only be myristoylated on lysine), NMT1 knockdown decreased myristoylation levels, which could be rescued by overexpression of either NMT1 or NMT2

• Biochemical approaches:

  • Using clickable fatty acid analogs (Alk12, Alk14) to metabolically label myristoylated proteins

  • Since NMT prefers myristoyl-CoA over palmitoyl-CoA, Alk12 labeling is more efficient than Alk14 and is abolished by pharmacological NMT inhibition

  • In vitro reactions with purified recombinant NMT1 and NMT2 to compare substrate preferences

These approaches, especially when used in combination, allow researchers to dissect the specific contributions of each NMT isoform to protein myristoylation in different cellular contexts.

How does the lysine myristoyltransferase activity of NMT2 differ from its canonical glycine myristoyltransferase activity?

The recently discovered lysine myristoyltransferase activity of NMT2 exhibits both similarities and differences compared to its canonical glycine myristoyltransferase activity:

The discovery of lysine myristoyltransferase activity significantly expands our understanding of NMT2's functional repertoire and its role in cellular regulation.

What methodologies are most effective for studying NMT2-substrate interactions?

Multiple complementary methodologies are employed to study NMT2-substrate interactions:

• Structural biology approaches:

  • X-ray crystallography to determine structures of NMT2 in complex with substrates and inhibitors

  • The 1.93 Å structure of NMT2 with a myristoylated lysine peptide demonstrates the molecular basis of lysine recognition

• Biochemical and cellular assays:

  • In vitro reactions with purified proteins to assess direct enzyme-substrate relationships

  • Co-immunoprecipitation (co-IP) experiments to detect physical interactions between NMT2 and potential substrates

  • Microscopy-based co-localization studies to examine spatial relationships

  • Metabolic labeling with clickable fatty acid analogs (Alk12, Alk14) followed by click chemistry and fluorescent detection

• Mass spectrometry-based approaches:

  • Identification of myristoylation sites in proteins isolated from cells

  • Quantitative chemical proteomics with NMT inhibition to identify substrate proteomes

  • 32P-NAD+ assays to detect lysine myristoylation through SIRT2-mediated removal

  • Thin-layer chromatography (TLC) to separate and identify myristoyl ADP-ribose products

These multifaceted approaches provide comprehensive insights into NMT2-substrate interactions at molecular, cellular, and functional levels.

What is the relationship between NMT2 and SIRT2 in regulating protein myristoylation?

NMT2 and SIRT2 form a dynamic regulatory system for protein lysine myristoylation:

• Complementary enzymatic activities:

  • NMT2 acts as the "writer" of lysine myristoylation, adding myristoyl groups to specific lysine residues

  • SIRT2, a NAD+-dependent deacylase, functions as the "eraser," removing myristoyl groups from lysine residues

  • This creates a reversible post-translational modification system

• Substrate preferences:

  • NMT2 preferentially modifies ARF6 in its GTP-bound (active) state

  • SIRT2 preferentially acts on ARF6 in its GDP-bound (inactive) state

  • This complementary preference couples the myristoylation-demyristoylation cycle to the GTPase cycle

• Experimental evidence:

  • 32P-NAD+ assays reveal that SIRT2 can remove myristoyl groups from ARF6 K3 but not G2

  • SIRT2 knockdown or inhibition increases ARF6 K3 myristoylation levels

  • Co-immunoprecipitation studies suggest a physical interaction between ARF6 and SIRT2

• Functional consequences:

  • This regulatory system affects ARF6 membrane association and trafficking

  • K3 myristoylation promotes ARF6 membrane localization during the GTPase cycle

  • SIRT2-mediated demyristoylation is required for efficient ARF6 activation

The table below summarizes the key differences in substrate preferences:

This NMT2-SIRT2 axis represents a sophisticated regulatory mechanism that can fine-tune protein function through reversible lysine myristoylation.

What experimental approaches can be used to identify novel substrates of NMT2?

Identifying novel NMT2 substrates requires sophisticated experimental strategies:

• Chemical proteomics approaches:

  • Metabolic labeling with clickable myristic acid analogs (Alk12, Alk14)

  • Bioorthogonal ligation to capture labeled proteins

  • Quantitative proteomics with and without NMT inhibition

  • This approach has identified >100 N-myristoylated proteins in human cells, with >95% being identified for the first time at endogenous levels

• Targeted substrate validation:

  • Creating mutants of potential substrates (G2A, K3R) to validate specific myristoylation sites

  • 32P-NAD+ assays with recombinant SIRT2 to detect lysine myristoylation

  • Thin-layer chromatography to separate and identify myristoyl ADP-ribose products

  • Microscopy-based approaches to assess membrane localization changes

• Sequence-based screening:

  • Screening for potential substrates with lysine at position 3 or 4

  • Insertion experiments show that adding one glycine between A2 and K3 in substrates is tolerated, while insertion of alanine significantly decreases myristoylation

These complementary approaches, when used in combination, provide a powerful toolkit for identifying and validating novel NMT2 substrates in various cellular contexts.

How does the crystal structure of NMT2 inform inhibitor design strategies?

The crystal structure of NMT2 provides valuable insights for inhibitor design:

• Key structural features:

  • Conserved substrate-binding pocket that accommodates both peptide substrates and myristoyl-CoA

  • Crystal structures of inhibitor-bound NMT1 and NMT2 demonstrate small-molecule inhibition through this pocket

  • The 1.93 Å structure showing a myristoylated lysine peptide product reveals critical binding interactions

• Structure-based design approaches:

  • Targeting the myristoyl-CoA binding site

  • Exploiting the peptide substrate binding pocket

  • Designing inhibitors that mimic the transition state of the reaction

• Quantitative assessment:

  • Dose-response relationships for inhibition of N-myristoylation can be determined for >70 substrates simultaneously across the proteome

  • This allows comprehensive evaluation of inhibitor specificity and efficacy

• Structural insights for specificity:

  • The flexibility observed in certain regions (e.g., adenosine-binding region) suggests potential for targeting dynamic structural elements

  • Comparison of NMT1 and NMT2 structures can guide the development of isoform-selective inhibitors

The availability of high-resolution structural data for NMT2, especially in complex with substrates and inhibitors, provides a strong foundation for rational drug design approaches targeting this enzyme.

How does NMT2 activity change during different cellular processes?

The activity and substrate specificity of NMT2 can vary during different cellular processes:

• Process-specific changes:

  • Global quantification of N-myristoylation during normal growth or apoptosis reveals distinct substrate profiles

  • Post-translational N-myristoylation may increase during apoptosis due to caspase-mediated protein cleavage, exposing new N-terminal glycines

• GTPase cycle-dependent activity:

  • NMT2 shows preference for active GTP-bound forms of ARF6

  • In vitro using purified proteins, reactions of NMT with the active ARF6 Q67L mutant produced more doubly myristoylated ARF6

  • Co-IP experiments showed that NMT2 preferentially binds to Q67L over T27N

  • NMT2 co-localizes with ARF6 Q67L better than with ARF6 T27N

• Regulatory significance:

  • The selectivity of NMT for ARF6-GTP and SIRT2 for inactive ARF6 accelerates ARF6 activation by avoiding a futile lysine myristoylation–demyristoylation cycle

  • This coupling mechanism ensures efficient coordination between protein modification and function

Understanding these dynamic changes in NMT2 activity provides insights into its role in cellular regulation and potential therapeutic targeting strategies.

What are the implications of NMT2 dysregulation in human diseases?

NMT2 dysregulation has been implicated in various human diseases:

• Therapeutic targeting potential:

  • NMT enzymes have been explored as therapeutic targets for infectious diseases:

    • Malaria

    • Sleeping sickness (trypanosomiasis)

    • Common cold

  • NMT inhibition has also been investigated for cancer treatment

• Pathophysiological roles:

  • N-myristoylation has been implicated in the development and progression of various human diseases

  • ARF6, a key substrate of NMT2, regulates essential trafficking and signaling pathways

  • Dysregulation of the NMT2/SIRT2-ARF6 regulatory axis may offer new ways to treat human diseases

• Potential mechanisms:

  • Changes in NMT2 expression or activity in disease states

  • Alterations in substrate availability or accessibility

  • Dysregulation of the dynamic myristoylation-demyristoylation cycle

Understanding the role of NMT2 in human diseases may reveal new therapeutic opportunities through targeted modulation of protein myristoylation.

What are the most promising strategies for developing selective NMT2 inhibitors?

Developing selective inhibitors for NMT2 versus NMT1 presents several challenges and opportunities:

• Current approaches:

  • Small-molecule inhibition through a conserved substrate-binding pocket has been demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2

  • Potent and specific human NMT inhibitors have been developed and used for quantitative chemical proteomics

• Selectivity considerations:

  • Despite structural similarities between NMT1 and NMT2, subtle differences in substrate binding regions may be exploited

  • The adenosine-binding region shows flexibility in NMT2 crystal structures, which might offer isoform-specific targeting opportunities

• Evaluation methods:

  • Quantitative dose-response for inhibition of N-myristoylation can be determined for >70 substrates simultaneously across the proteome

  • This allows comprehensive assessment of inhibitor specificity against both NMT isoforms

• Therapeutic potential:

  • NMT inhibitors have been explored for treating malaria, sleeping sickness, common cold, and cancer

  • Understanding the distinct roles of NMT1 versus NMT2 in different contexts will be crucial for developing therapeutically useful selective inhibitors

Further structural and functional studies of both NMT isoforms will be essential for developing truly selective inhibitors with therapeutic potential.

How can researchers optimize experimental conditions for studying NMT2 in vitro?

Optimizing experimental conditions for studying NMT2 in vitro requires careful consideration of several factors:

By optimizing these experimental parameters, researchers can effectively study NMT2 activity, substrate specificity, and inhibitor interactions in vitro.

What are the key unresolved questions about NMT2?

Despite significant advances in our understanding of NMT2, several important questions remain:

• Substrate selectivity mechanisms:

  • How do NMT1 and NMT2 achieve differential substrate specificity despite structural similarities?

  • What structural or cellular factors determine whether a protein undergoes glycine or lysine myristoylation?

  • Are there other substrates regulated by the lysine myristoyltransferase function of NMT beyond ARF6?

• Regulatory networks:

  • How is NMT2 activity itself regulated at transcriptional, translational, and post-translational levels?

  • Are there other deacylases besides SIRT2 that can remove lysine myristoylation?

  • What is the full extent of the NMT2/SIRT2-ARF6 regulatory axis in cellular function?

• Physiological significance:

  • What is the relative abundance and physiological importance of lysine versus glycine myristoylation in different cell types and tissues?

  • How does lysine myristoylation contribute to the unique plasma membrane targeting of ARF6 compared to other ARFs?

  • What other cellular processes might be regulated by dynamic lysine myristoylation?

Addressing these questions will significantly advance our understanding of NMT2 biology and its therapeutic potential.