Clinical Applications of an AMH Therapeutic

AMH induces a BMP-Like Smad activation however the target genes are distinct from other BMPs
In the male fetus, AMH triggers apoptosis of the Müllerian duct mesenchyme
Signaling in the ovary by granulosa-expressed AMH functions through a feedback inhibition mechanism to limit primordial follicle selection and maturation
- This mechanism is useful for clinically regulating fertility
Recent studies have successfully applied AAV-delivered AMH to cats for nonsurgical sterilization

A BMP Family Outlier

Of the ~30 genes in the TGFβ family, AMH is a distinct outlier, often grouped with inhα or the GDNF family member, GDF15
The AMH prodomain is the largest in the family and cannot be confidently aligned with any other sequence
While the AMH growth factor (GF) domain has a negligible effect on cell signaling in vitro, assays which apply AMH ex vivo show a profound activity of the prodomain in enhancing the activity of the GF
We found that the AMH prodomain consists of two separate subdomains: an AMH-specific Helical binding domain (HBD) which engages the GF and a TGFβ propeptide domain (TPD) which covalently tethers a prodomain dimer
- The TPD is flanked by an N-terminal extension and separated from the HBD by a proline-rich linker

By mapping the gene structure of AMH among all chordate species, an addition to human exon 5 was identified to contain the HBD
- This suggests a gain-of-function recombination that triggered the divergence of the AMH gene
- No homologous proteins can be found for HBD by sequence or structure
The core of the TPD, although it has lost its GF binding function, maintains a canonical TGFβ propeptide architecture, consisting of an 8-sheet jelly roll fold with interspersed helices
When compared to TGFβ1, the TPD shares a similar position for one of its disulfide bonds, however the position of the jelly roll fold is inverted

Cryo-EM with mAb 6E11 was used to determine a 3.2Å structure of the GF:HBD complex
The HBD consists of a four helix bundle and “binding belt” which engage the type I and type II receptor binding sites on the AMH GF
Flexibility of the procomplex limited its resolution, and a large region between the belt and Cα3 is unresolved

The AMH prodomain contains 5 cysteine residues
- Cys55 is the strongest dimerizing cysteine with Cys55 of the other chain
- Cys103 forms an intramolecular disulfide bond with Cys188
- Cys241 is a weaker dimerizing cysteine with Cys241 of the other chain
- Cys411 is unpaired and may contribute to oligomerization
Mutations targeting these cysteine residues alter the molecular state of AMH yet retain ex vivo bioactivity
Negative stain EM studies confirm that the AMH prodomain has 2 subdomains and that the TPD is flexibly tethered to the HBD
Small angle X-ray scattering and MD simulation support a flexible, semi-compact two domain structure of the AMH procomplex
Disease-associated mutations are spread throughout the prodomain
- Persistent Müllerian duct syndrome mutations cluster in the TPD, presenting a loss-of-function phenotype
- Polycystic ovary syndrome mutations have an unclear phenotype, but they are scattered across all components

3D variability analysis of the cryo-EM data reveals significant heterogeneity within the fab-bound procomplex
- The majority of particles have weak or absent prodomain density
When in solution (vitreous ice), the prodomain exhibits a range of motion between 8Å to 12Å, while the resolved structure is in an intermediate position
- This motion is achieved by “sliding” of nonpolar residues in the prodomain helices against the other helices or the GF fingers
Hydrogen-deuterium exchange MS highlights the conformational variability within the AMH procomplex
- The TPD is moderately dynamic: loops are dynamic while the core, especially around Cys241, is stable
- The N-terminus and linker are highly flexible: early residues in the linker are rapidly dynamic while later residues are fully unstructured
- The HBD is very dynamic: Cα1-4 helices and belt show slow exchange kinetics while α5 is stable
- The Gf is moderately dynamic: finger 1/2 has slow exchange kinetics indicating a dynamic conformation
MD simulations of the Procomplex demonstrate the broad flexibility of the prodomain relative to the GF core
- The binding belt and α5 are the most stable

Bivalent binding of AMHR2 to the AMH GF induces a conformational shift within the GF
When comparing the Cryo-EM structure with the AMHR2-bound crystal structure, the GF is in an open and extended state when bound to the HBD in solution (vitreous ice)
- Transitioning between the states involved compression of the GF by ~15Å and a 38° rotation of the fingers
Both open and closed GF conformations are stabilized by extensive hydrogen bonds between finger 1/2 and 3/4 as well as within finger 3/4
- Leu478 is stabilized by Arg302 in the prodomain or Arg97 in AMHR2
- The open GF structure is the most open and extended GF among every solved GF structure in the PDB
MD simulations show that while the open GF is much less stable than the closed GF, there is no transition between them even when unbound, suggesting AMHR2 is required for the conformation to switch

Before this study, it was unknown how AMHR2 can displace the prodomain from the GF despite having a much weaker affinity for the GF
Binding energies calculated by steered MD and umbrella sampling show that while the full procomplex binds the GF very strongly, the helical bundle component and AMHR2 have more comparable values
AMHR2 uses fewer, more stable residues to engage the GF while the HBD spreads its binding motif across many residues, many of which are highly flexible
NMR binding studies show that even when bound to AMHR2 in solution, AMH is not capable of binding ALK2, likely due to its open conformation
The dynamic HBD is further assisted by the the TPD, which enhances GF affinity by avidity for a bivalent HBD:GF interaction
Thus, the displacement and signaling mechanism is as follows:
- Step 1: two helical bundles, whose binding is transiently weakened by internal flexibility, are independently displaced by two AMHR2 molecules while the binding belt remains in complex
- Step 2: Bivalent binding of AMHR2 (only when surface-bound) provides the energy to flip the plane of Val477 and Leu478 in the GF fingers, remodeling both the AMHR2 and belt binding sites to close and lock the GF conformation about Glu474 on one chain and Lys512/Arg/516 on the other chain
- Step 3: the remodeled GF is now able to bind to ALK2/3/6 using the same residues which it used for the belt