Posttranslational modifications remodel proteome-wide ligandability

Li, W., Wei, Q., Llanos, M. et al.

Nat Chem Biol (2026). https://doi.org/10.1038/s41589-026-02216-y

Posttranslational modifications (PTMs) vastly expand the diversity of the human proteome, dynamically reshaping protein activity, interactions and localization in response to environmental, pharmacologic and disease-associated cues. However, their proteome-wide impact on small-molecule recognition—and, thus, druggability—remains largely unexplored. Here we present a chemical proteomic strategy to delineate how PTM states remodel protein ligandability in human cells. Using broad-spectrum photoaffinity probes, we identified more than 400 functionally diverse proteins whose ability to engage small molecules is impacted by phosphorylation or N-linked glycosylation status. Integrating binding site mapping with structural analyses reveals a diverse array of PTM-dependent pockets. Among these, we discovered that the phosphorylation status of common oncogenic KRAS mutants impacts the action of small molecules, including clinically approved inhibitors. These findings illuminate a previously underappreciated layer of proteome plasticity governed by PTMs and highlight opportunities to develop chemical probes that selectively target proteins in defined modification states.

논문 요약

Posttranslational modifications remodel proteome-wide ligandability] – Original article

-          PTM(번역후변형)은 인간 단백질체를 크게 확장하며 단백질 활성·상호작용·위치를 조절함. 그러나 PTM이 소분자 인식(druggability)에 미치는 영향에 관한 연구는 부족한 실정임. 기존 ABPP 방법은 반응성 잔기(주로 cysteine)에 국한되고, 세포 용해물(lysate)에서 수행돼 native 상호작용이 교란되는 한계가 있었음.

-          방법: FFF(fully-functionalized fragment) photoaffinity probe 기반 화학단백질체학을 살아있는 세포에서 수행. 각 probe는 결합용 fragment + diazirine(UV crosslink) + alkyne(click)으로 구성. 8종 probe(P1~P6, 이 중 P5·P6는 (R)/(S) 거울상 쌍) 사용.

-          두 PTM을 조작해 상태를 비교:

①      인산화 — MDA-MB-231 세포에 STR(staurosporine, pan-kinase 억제제) 처리

②      당화 — HEK293T 세포에 Tuni(tunicamycin, N-당화 억제제) 처리

-          세포에 probe 처리 → UV crosslink → click으로 biotin 태그 → 농축 → TMT 표지 → 질량분석 정량.

주요 결과

1.       STR 처리로 인산화 1,911곳 감소·542곳 증가. probe 결합이 PTM 상태에 따라 유의하게 변한 단백질을 (인산화 235개, 당화 225개) 확인. 이 결합 변화는 단백질 양(abundance) 변화와 독립적임.

2.       다양한 단백질군 확인: 효소·이온채널뿐 아니라 전통적으로 어려운 표적인 전사인자·후성유전 조절자까지 포함. 대부분(~78%)은 잘 알려진 리간드가 없는 단백질.

3.       검증JAK1(인산화로 결합 감소), ATP6V1E1(P6에서 증가), EDEM3·CDH2(당화로 증가) 등을 chemoprecipitation(ChP) western blot으로 재확인. Input은 고정, ChP만 변동 → 결합력 변화 입증.

4.       두 가지 메커니즘 확인 결합 부위가 PTM 자리 근처(대부분 10Å 이내)에 몰림.

①      Proximal(직접): PTM이 pocket을 직접 막거나 열음 — HSPB1(S82), CTSD(S37), GABARAPL2(S87/88), EphA2(Y772), NPC2(N58, 콜레스테롤 pocket)

②      Distal(간접): PTM이 멀리서 단백질 복합체를 바꿔 원격으로 pocket 노출 — LAMTOR2(S26 인산화가 LAMTOR1 결합을 바꿔 'neo-pocket' 생성)

-          돌연변이(GABARAPL2 S87/88A, NPC2 N58A)로 인과관계 유전학적 증명.

5.       KRAS 임상 사례 : KRAS의 Y32/Y64 인산화가 switch-II pocket 리간드 결합을 조절. 인산화가 SOS1 결합을 방해해 GDP-결합 형태(약물이 선호하는 형태) 비율을 높임. 이것이 승인·개발 중인 KRAS 억제제(BI-2865, AMG-510, MRTX-849 등)의 결합·효능에 영향. SPR로는 친화도 차이가 없어(내성 아닌 형태 변화), CETSA·nucleotide exchange assay로 기전 규명함.

결론

·         PTM 상태는 단백질 druggability를 조절할 수 있음. 이것은 질병 관련 proteoform의 차별적 druggability를 드러내며, 특정 변형 상태의 단백질만 선택적으로 겨냥하는 화학 probe·치료제 설계 가능성을 제시함.

Figure 5. KRAS phosphorylation status alters druggability.

a,b, Proteomic profile (a) and chemoprecipitation (ChP) validation (b) of P3 binding to KRAS^G13D after STR treatment (250 nM, 3 hours, n = 2). c, Docked KRAS−P3 interaction near the SII pocket (PDB: 8B00), with P3-labeled residues shown in blue, phosphorylation sites (Y32 and Y64) in red, P3 in yellow and GDP in cyan. d, Dose-dependent P3 competition by the pan-KRAS inhibitor BI-2865. e, KRAS phosphorylation level decreased after STR (250 nM, 3 hours) treatment. HA-tagged KRAS was immunoprecipitated, and phosphorylation status was assessed by immunoblotting using a pan-phosphotyrosine antibody. f, ChP shows reduced P3 binding to Y32F and Y64F mutants of KRAS^G13D. g, Increased P3 engagement with KRAS^G13D upon SHP2 inhibition by SHP099 (500 nM, 30 minutes). h, CETSA reveals decreased KRAS stabilization by BI-2865 in the presence of STR, whereas SHP099 has no observable effect. Data are presented as mean with 95% confidence intervals for curve fits. i, RBD assay shows that STR diminishes the ability of KRAS-OFF inhibitors to disrupt the active (GTP-bound) KRAS−RAF interaction across multiple KRAS-mutant cell lines, whereas SHP099 restores the effect. Immunoprecipitation (IP) reveals that STR enhances KRAS−CYPA ternary complex formation in the presence of the KRAS-ON state inhibitor RMC-7977. j, Total pERK (T202/Y204) levels assessed by immunoblotting in cells co-treated with dasatinib (1 µM) and indicated KRAS inhibitors restore pERK across various KRAS mutations in multiple cell lines. k,l, Co-IP shows that the KRAS−SOS1 interaction is enhanced by Y32F/Y64F mutation (k) and STR treatment (l) but attenuated by SHP099 (l). m, In vitro fluorescence-based nucleotide exchange assay demonstrates that phosphorylation impedes SOS1-mediated GDP release, as mono-phosphorylated (pKRAS) and di-phosphorylated (ppKRAS) forms exhibit slower exchange kinetics compared to non-phosphorylated KRAS (KRAS:SOS 10:1). n, Quantification of the fold change in exchange rates (+SOS1/−SOS1). Proteomics and immunoblots represent the mean of two (data points overlaid) or three (±s.d.) independent biological replicates. See Supplementary Tables 2 and 13 for related datasets. CI, confidence interval; Tm, melting temperature; KRASi, KRAS inhibitor; min, minutes; WT, wild-type.