The DOT1L–ubiquitin and DOT1L–acidic-patch interfaces: two non-catalytic surfaces that MV4;11 cells cannot do without
also called DOT1L ubiquitin-binding surface · DOT1L acidic patch interface · Dot1L-H2BK120ub nucleosome interface
DOT1L grips the nucleosome in two places that have nothing to do with the chemistry it performs: the ubiquitin tag on H2B, and a negatively charged pocket on the nucleosome surface. Breaking either grip stops a KMT2A-fusion leukaemia cell growing, which makes them targets that are not the drug site that already failed in the clinic.
The same thing, for a scientist
Four independent 2019 cryo-EM studies resolved DOT1L on the H2BK120-ubiquitinated nucleosome, with structures in the PDB at up to 2.96 Å [E2]. The picture is consistent: DOT1L is anchored by the conjugated ubiquitin and by the H2A/H2B **acidic patch**, with the histone H4 N-terminal tail contributing to positioning; these contacts induce a conformational change in the globular core of H3 that reorients K79 into the active site [E1]. Spangler et al. then made the functional test that matters for this graph: structure-guided DOT1L mutations disrupting ubiquitin binding, and separately disrupting acidic-patch binding, each **abolish proliferation of MV4;11**, a KMT2A::AFF1 line — and the biophysics shows ubiquitin and cofactor binding drive conformational changes that promote *productive* nucleosome binding rather than simply more binding [E3]. That is a genetic proof of principle for targeting a surface rather than the SAM pocket. Pinometostat, the SAM-competitive inhibitor this graph already carries, reduced H3K79 methylation in patients and produced only limited clinical responses; whatever explains that failure, it is not a reason to abandon DOT1L, and these interfaces are a mechanistically distinct handle on the same enzyme.
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The ubiquitin is attached to H2B K120 and contacts DOT1L directly; a second anchor is the acidic patch on the H2A/H2B surface.
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Compare the poised state 6NOG with the active states 6NQA and 6NJ9: the conformational change between them is the mechanism by which the ubiquitin switches the enzyme on.
The structures, verified against the PDB rather than recalled
| PDB | Resolution (Å) | Title as deposited |
|---|---|---|
| 6NJ9 | 2.96 | Active state Dot1L bound to the H2B-Ubiquitinated nucleosome, 2-to-1 complex |
| 6NQA | 3.54 | Active state Dot1L bound to the H2B-Ubiquitinated nucleosome, 1-to-1 complex |
| 6NOG | 3.90 | Poised-state Dot1L bound to the H2B-Ubiquitinated nucleosome |
| 6O96 | 3.50 | Dot1L bound to the H2BK120 Ubiquitinated nucleosome |
| 6JMA | 6.80 | cryo-EM structure of DOT1L bound to H2B ubiquitinated nucleosome |
| 6JM9 | 7.30 | cryo-EM structure of DOT1L bound to unmodified nucleosome (the control) |
Every identifier, resolution and title in this table was fetched from the RCSB data API for this entry on 2026-09-19, not recalled 2. The existence of a poised state and an active state in the same series (6NOG vs 6NQA/6NJ9) is the structural expression of the conformational-change mechanism, and a poised state that a small molecule could trap is the most concrete drug-discovery proposition in this beat.
No interface residue numbers are asserted in this entry. The papers define the contacts, but this beat read abstracts rather than full texts and the project's standing rule is that a residue number you have not read is not a residue number. data.interface_residues_a is therefore empty by choice, not by oversight.
Why this is different from the drug that failed
Pinometostat is SAM-competitive, binds in the catalytic pocket, and achieved pharmacodynamic H3K79 methylation reduction in a phase 1 trial with limited clinical responses (see mechanism-dot1l-h3k79-methylation and drug-pinometostat).
The surfaces described here are different in three specific ways:
- They are not the SAM site, so cross-resistance with pinometostat is not automatic. Treatment-emergent pinometostat resistance was generated in five KMT2A-rearranged lines without DOT1L target mutation (the graph's DOT1L entry, E8 there), which is consistent with resistance arising outside the catalytic pocket.
- Blocking them is not the same as blocking catalysis. 3's mutants fail to bind productively at all; the enzyme is not merely slowed. There is a published argument that DOT1L has methyltransferase-independent functions — it promotes H2B ubiquitination through its N-terminus without catalysis in yeast — and a surface-directed agent that prevents nucleosome engagement would remove more of DOT1L's function than a SAM-site inhibitor does.
- The acidic patch is shared. Many chromatin factors bind the H2A/H2B acidic patch, which is simultaneously the reason it is chemically approachable and the reason an acidic-patch-directed agent would be promiscuous. That is a serious liability and it is in
bite_back.
None of this is a compound. There is no reported small molecule against either surface, and both are large, shallow protein-protein interfaces of the kind that have defeated many campaigns.
| partner a | gene-dot1l |
|---|---|
| partner b | mark-h2bk120ub |
| interface residues note | Not asserted. The structures define the contacts but this beat read abstracts, not full texts, and the project's rule is that an unread residue number is not a residue number. |
| motif b | ubiquitin conjugated to histone H2B lysine 120; second anchor at the H2A/H2B acidic patch; histone H4 N-terminal tail contributes to positioning |
| functional validation | structure-guided DOT1L ubiquitin-binding and acidic-patch mutants each abolish MV4;11 (KMT2A::AFF1) proliferation |
| druggability | Two large, shallow protein-protein/protein-nucleosome surfaces with no reported chemical matter. The strongest argument for tractability is the existence of distinct poised and active conformations (6NOG vs 6NQA/6NJ9), because a conformational equilibrium can in principle be trapped. The strongest argument against is that the acidic patch is a shared docking site for many chromatin factors. |
interface residues a
[]
structures
[ "6NJ9", "6NQA", "6NOG", "6O96", "6JMA", "6JM9" ]
structure resolutions angstrom — the numbers behind the chart
{
"6NJ9": 2.96,
"6NQA": 3.54,
"6NOG": 3.9,
"6O96": 3.5,
"6JMA": 6.8,
"6JM9": 7.3
}ligands
[]
- The nucleosome acidic patch is bound by a large number of chromatin proteins, so an acidic-patch-directed molecule would be promiscuous by construction and its toxicity would be chromatin-wide.
- DOT1L has already failed clinically once in this disease at a different site on the same enzyme; the burden of proof that a new site changes the outcome sits with the new site.
- A separation-of-function mutant that kills MV4;11 is not the same as a drug that kills MV4;11; mutant rescue removes 100% of the interaction from 100% of the protein, which no inhibitor achieves.
- If H2BK34 ubiquitination can position DOT1L productively without contacting it (see mark-h2bk34ub), an agent aimed at the DOT1L-ubiquitin contact may be bypassed by the K34 route.
- Would blocking the DOT1L-ubiquitin interface avoid pinometostat cross-resistance? The five pinometostat-resistant lines already exist in the literature and have no DOT1L mutation; testing the ubiquitin-binding mutant in that background is a direct experiment.
- Is the acidic-patch contact separable from the ubiquitin contact pharmacologically? If not, any agent hitting the acidic patch will hit dozens of other chromatin factors and will not be a DOT1L drug.
- Does a DOT1L allele that cannot bind ubiquitin still support the methyltransferase-independent functions attributed to DOT1L's N-terminus? If it does, a surface-directed agent and a degrader are not equivalent.
- Has anyone attempted a fragment or macrocycle campaign against the poised-state conformation captured in 6NOG? Nothing in this beat's reading says so.
- E1established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.paper
Cryo-EM of Dot1L bound to the ubiquitinated nucleosome shows how H2B-Ub stimulates Dot1L activity and reveals a role for the histone H4 tail in positioning Dot1L; contacts mediated by Dot1L and the H4 tail induce a conformational change in the globular core of histone H3 that reorients K79 from an inaccessible position into the active site primed for catalysis.
Mechanism of Cross-talk between H2B Ubiquitination and H3 Methylation by Dot1L. · Worden EJ, Hoffmann NA, Hicks CW, Wolberger C · Cell 2019What this evidence cannot show
Abstract read in full; full text not read. Reconstituted complexes; no residue-level detail is taken from this abstract and none is asserted here.
Evidence that argues against this (1)
- A cryo-EM structure of DOT1L on a ubiquitinated nucleosome shows how the mark stimulates the enzyme, which is a mechanism of stimulation and not a measure of how much of DOT1L's cellular activity depends on it. The structure is also of a defined reconstituted nucleosome rather than chromatin. Cell 2019
- E2established confidence 5 of 5: Multiple independent lines of evidence; treated as settled in the field.database
The Protein Data Bank contains experimentally determined cryo-EM structures of DOT1L bound to the H2B-ubiquitinated nucleosome: 6NJ9 (2.96 A, active state, 2-to-1 complex), 6NQA (3.54 A, active state, 1-to-1), 6NOG (3.90 A, poised state), 6O96 (3.50 A, H2BK120-ubiquitinated nucleosome), 6JMA (6.80 A), with 6JM9 (7.30 A) the corresponding unmodified-nucleosome complex.
RCSB Protein Data Bank entries 6NJ9, 6NQA, 6NOG, 6O96, 6JMA, 6JM9; identifiers, resolutions and deposited titles retrieved from the RCSB data API on 2026-09-19 · RCSB Protein Data Bank; retrieval performed by this project · Database 2026What this evidence cannot show
The list comes from a full-text search of the PDB for 'DOT1L ubiquitinated nucleosome' returning six entries; it is not guaranteed exhaustive, and entries whose titles do not contain those words would be missed. Resolutions are the combined values reported by the API. This beat did not inspect the coordinates or validation reports.
Evidence that argues against this (1)
- Listing PDB accessions establishes that structures exist and does not adjudicate between them. Independent cryo-EM structures of this complex differ in resolution and in which contacts are modelled, so citing the set as one fact hides the disagreements a reader would need in order to use the coordinates. Cell 2019
- E3strong confidence 4 of 5: Replicated in peer-reviewed work by more than one group.paper
Bulk and single-molecule biophysical measurements combined with cancer cell biology show that ubiquitin and cofactor binding drive conformational changes that stimulate DOT1L activity, and structure-guided mutations demonstrate that ubiquitin binding and nucleosome acidic-patch binding by DOT1L are each required for cell proliferation in the MV4;11 leukaemia model, providing proof of principle for MLL-targeted therapeutic strategies.
DOT1L activity in leukemia cells requires interaction with ubiquitylated H2B that promotes productive nucleosome binding. · Spangler CJ, Yadav SP, Li D, Geil CN, Smith CB, Wang GG, Lee TH, McGinty RK · Cell Rep 2022What this evidence cannot show
Abstract read in full; full text not read. One cell line. A rescue-with-mutant design can fail for reasons other than the intended interface - misfolding, altered stability, altered expression - and the abstract does not say which controls excluded those. 'Proof of principle for MLL targeted therapeutic strategies' is the authors' framing; no compound exists.
Evidence that argues against this (1)
- The clinical precedent for targeting DOT1L is discouraging: pinometostat achieved pharmacodynamic H3K79 methylation reduction in a phase 1 trial of 51 adults with limited responses, and treatment-emergent resistance arose in five KMT2A-rearranged lines without DOT1L mutation. Both facts are already carried in this graph's mechanism-dot1l-h3k79-methylation entry with their own citations. A different binding site does not by itself repair a target whose inhibition has already proved insufficient. Oncofusion Knowledge Graph et al. 2026
- ← related toThe trans-histone crosstalk H2Bub → DOT1L → H3K79me: modulatory for me1, effectively required for me2/me3
Counter-evidence (Zhou 2016, PMID 26830124): photocrosslinking in semisynthetic designer chromatin places the functional hotspot on ubiquitin against the H2A N-terminus and describes H2B-Ub corralling hDot1L into a productive orientation - a repositioning model competing with the allosteric H3-core reorientation this entry asserted, and obtained with the human enzyme
two non-catalytic surfaces of DOT1L: the ubiquitin contact and the acidic-patch contact
DOT1L binds the ubiquitin conjugated to H2B K120
- related to →Pinometostat (EPZ-5676) — the DOT1L inhibitor, and the most instructive failure in this field
a non-SAM binding site on the same enzyme that failed clinically at the SAM site
NF-YB carries a Lys138 ubiquitination reported equivalent to H2BK120ub, the mark that licenses DOT1L H3K79 methylation; the residue is on NF-YB, not NFYC
a second ubiquitin-like ligand for DOT1L's UIM; potential competitor of H2Bub