Structures of the HER2-HER3-NRG1β complex �reveal a dynamic dimer interface:��Nature 600 (7888) 339-43 (2021)��Auburn University Cancer Research Journal Club�January 23, 2023

David J. Riese II, Ph.D.

Professor, Auburn University Harrison College of Pharmacy

Senior Scientist, UAB O’Neal Comprehensive Cancer Center

Link and QR Code to Presentation

AU Cancer Research Journal Club Slide 2

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• Questions, methods, and experimental results
• Conclusions and future directions

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AU Cancer Research Journal Club Slide 3

Presentation outline

• Overarching goal(s)/hypothesis(es)
• How does the structure of the HER2-HER3-NRG1β complex explain stimulation of HER2-HER3 heterodimers with ligand, the activity of the gain-of-function (oncogenic) HER2 S310F mutant, and antagonism of HER2 signaling by the anti-HER2 mAb drugs trastuzumab and pertuzumab?
• Background
• Questions, methods, and experimental results
• Conclusions and future directions

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AU Cancer Research Journal Club Slide 4

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• What was previously known about the structure and function of ERBB/HER receptor tyrosine kinases?
• Questions, methods, and experimental results
• Conclusions and future directions

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AU Cancer Research Journal Club Slide 5

Structure and function of ERBB/HER receptors I

• Transmembrane proteins
• Extracellular domain exists in an equilibrium between the open and closed conformations
• The open conformation exposes dimerization motifs
• Ligand binding stabilizes the open conformation

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AU Cancer Research Journal Club Slide 6

Lauren Lucas’ dissertation defense seminar, January 2023

Structure and function of ERBB/HER receptors II

• Symmetrical homo- and hetero-dimerization of extracellular domains
• Asymmetrical dimerization of intracellular domains enables cross-phosphorylation of tyrosine residues
• How is HER2 regulated?
• Why do multiple ligands bind to the same receptor?

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AU Cancer Research Journal Club Slide 7

Lauren Lucas’ dissertation defense seminar, January 2023

Different ligands for HER3 are functionally distinct: �The Q43L mutant of NRG2β competitively antagonizes �wild-type NRG2β at HER2-HER3 heterodimers

• NRG2β is a full agonist of HER3
• The Q43L mutant of NRG2β and wild-type NRG2β bind with equal affinity to HER3
• But NRG2β/Q43L does not stimulate phosphorylation on as many HER3 tyrosine residues as wild-type NRG2β and does not stimulate the coupling of HER2-HER3 heterodimers to cell proliferation
• What is the mechanism of this specificity?

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AU Cancer Research Journal Club Slide 8

Biochem J 443 (01) 133-44 (2012)

Different figands for EGFR are functionally distinct, too: �AR and EREG are full agonists at the EGFR, whereas EGF is a partial agonist

• AR and EREG stimulate greater cell proliferation than EGF
• EGF stimulates greater phosphorylation of EGFR Y1045 than AR.
• The EGFR Y1045F mutant enables EGF to stimulate greater cell proliferation

AU Cancer Research Journal Club Slide 9

Growth Factors 30 (02) 107-16 (2012)

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• Questions, methods, and experimental results
• How does HER2-HER3 heterodimerization enable ligand-stimulation of HER2-HER3 signaling?
• Conclusions and future directions

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AU Cancer Research Journal Club Slide 10

Cryo-EM reveals the structure of the HER2/HER3/NRG1β complex

• Methodology
• Results:
• The HER2 extracellular domain (ECD) is in the open conformation
• NRG1β stabilizes the HER3 ECD in the open conformation
• The open conformation of the HER2 and HER3 ECDs enables their heterodimerization that resembles canonical ligand-induced HER dimerization
• HER3 dimerization arm is unresolved
• The heterogenous conformation may enable ligand-specific conformational isoformers (conformers) and ligand-specific signaling
• The heterogenous structure reduces the affinity of HER3 for HER2, thereby reducing ligand-dependent and -independent (stochastic) HER2-HER3 dimerization and signaling

AU Cancer Research Journal Club Slide 11

Nature 600 (7888) 339-43 (2021)

The HER2 ECD exists in the open conformation but does not contain a pocket for the dimerization arm of the HER3 ECD

• The HER2 ECD is in the open conformation
• The HER3 ECD, when bound to NRG1b, is stabilized in the open conformation and contains a ”closed” pocket for tightly binding the dimerization motif of the HER2 ECD
• The HER2 ECD, despite being in the open conformation, contains an “open” pocket that does not tightly bind the dimerization motif of the HER3 ECD – thus, HER3 ECD dimerization motif is relatively unstructured and the HER2-HER3 heterodimer is weakly bound
• The difference in the conformation of the HER2 and HER3 ECDs resembles the difference in the conformation of the EGFR bound by EREG and EGFR bound by EGF
• EGFR bound with EREG forms a “partially closed” pocket for the EGFR dimerization arm and EGFR bound with EGF forms a “closed” pocket for the EGFR dimerization arm – thus, EGFR dimers caused by EGF binding are more stable than EGFR dimers caused by EREG binding
• Remember that EREG does not stimulate EGFR phosphorylation at Y1045, but EGF does – accounting for the functional differences between EREG and EGF
• This suggests that phosphorylation of Y1045 occurs with slow kinetics following stimulation with EREG, but with fast kinetics following stimulation with EGF. Thus, EGFR dimers caused by EREG binding are not stable enough to permit phosphorylation of Y1045, thereby preventing phosphorylation-dependent ubiquitination of EGFR by Cbl and EGFR downregulation

AU Cancer Research Journal Club Slide 12

Nature 600 (7888) 339-43 (2021)

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• Questions, methods, and experimental results
• How does HER2-HER3 heterodimerization enable ligand-stimulation of HER2-HER3 signaling?
• Does HER2-HER3 heterodimerization account for the oncogenic (gain-of-function) activity of the HER2 S310F mutant?
• Conclusions and future directions

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AU Cancer Research Journal Club Slide 13

The HER2 S310F mutant fosters HER2-HER3 heterodimerization by increasing HER2 binding to the HER3 dimerization arm

• The HER2 S310F mutation resides in the “open” dimerization arm binding pocket of the HER2 EC subdomain IV
• HER2 F310 forms a pi-pi interaction with Y265 of the HER3 dimerization arm
• Increases HER2-HER3 heterodimerization and signaling
• Physical interaction also facilitated by polar interactions of HER2 F310 with the backbone atoms of HER3 F291 and C311

AU Cancer Research Journal Club Slide 14

Nature 600 (7888) 339-43 (2021)

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• Questions, methods, and experimental results
• How does HER2-HER3 heterodimerization enable ligand-stimulation of HER2-HER3 signaling?
• Does HER2-HER3 heterodimerization account for the oncogenic (gain-of-function) activity of the HER2 S310F mutant?
• How do the anti-HER2 mAb drugs trastuzmab and pertuzumab possess complementary anti-tumor activities?
• Is it related to the fact trastuzumab inhibits signaling by HER2 homodimers, whereas the anti-HER2 mAb drug pertuzumab inhibits signaling by HER2-HER3 heterodimers?
• Can the structure of HER2-HER3 heterodimers with pertuzumab or trastuzumab yield mechanistic insights?
• Conclusions and future directions

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AU Cancer Research Journal Club Slide 15

The anti-HER2 mAb trastuzumab does not block HER2-HER3 heterodimerization but the anti-HER2 mAb pertuzumab does

• Trastuzumab binds HER2 EC subdomain IV, but does not block formation of the HER2-HER3-NRG1β ternary complex
• Pertuzumab binds HER2 EC subdomain II and blocks formation of the HER2-HER3-NRG1β ternary complex
• This inhibition can be overcome by the S310F mutation of the dimerization arm binding pocket of HER2 EC subdomain II

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AU Cancer Research Journal Club Slide 16

Nature 600 (7888) 339-43 (2021)

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• Questions, methods, and experimental results
• Conclusions I
• The HER3 dimerization arm is relatively unstructured
• The HER2 ECD is in the open conformation, but is not ideally structed to facilitate HER2-HER3 heterodimerization
• Limit stochastic HER2-HER3 heterodimerization and ligand-independent signaling
• Enable ligand-specific signaling?
• Future directions

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AU Cancer Research Journal Club Slide 17

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• Questions, methods, and experimental results
• Conclusions II
• The HER2 S310F mutation increases the interaction of the HER2 dimerization arm binding pocket with the HER3 dimerization arm and signaling by the HER2-HER3 heterodimer
• HER2 F310 interacts with HER3 Y265, F291, and C310
• Future directions

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AU Cancer Research Journal Club Slide 18

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• Questions, methods, and experimental results
• Conclusions III
• Trastuzumab can bind to HER2-HER3-NRG1β complexes, but pertuzumab cannot
• Consistent with the observation that trastuzumab does not inhibit signaling by HER2-HER3 heterodimers
• Consistent with the observation that pertuzumab does inhibit signaling by HER2-HER3 heterodimers
• Pertuzumab can bind to HER2-HER3-NRG1β complexes in the context of the HER2 S310F mutation
• Future directions

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AU Cancer Research Journal Club Slide 19

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• Questions, methods, and experimental results
• Conclusions
• Future directions I
• How do full and partial agonists of HER3 cause differences in the structure of HER2-HER3 heterodimers? Specifically, do full and partial agonists cause differences in the conformation of the HER3 dimerization arm binding pocket that binds the HER2 dimerization arm?
• Does the HER2 S310F mutation increase HER2 homodimerization? Does the HER2 S310F mutation increase HER2-HER4 heterodimerization?
• Is the dimerization arm of HER4 unstructured? Do some of the oncogenic (gain-of-function) mutants of HER4 reside in this dimerization arm and do they cause increased HER4-HER2 or HER4-EGFR heterodimerization? Do some of the oncogenic mutants of HER4 reside in the HER4 heterodimerization arm binding pocket and do they cause increased HER4-HER2 or HER4-EGFR heterodimerization?

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AU Cancer Research Journal Club Slide 20

Presentation outline

• Overarching goal(s)/hypothesis(es)
• Background
• Questions, methods, and experimental results
• Conclusions
• Future directions II
• Do full and partial agonists of HER4 cause differences in the structure of HER2-HER4 heterodimers? Specifically, do full and partial agonists cause differences in the conformation of the HER4 dimerization arm binding pocket that binds the HER2 dimerization arm?
• Are different tyrosine residues of EGFR phosphorylated with different kinetics? Specifically, is EGFR Tyr1045 phosphorylated with slow kinetics following stimulation with EREG but with rapid kinetics following stimulation with EGF?
• Do the differences in the effects of trastuzumab and pertuzumab on HER2 signaling account for the differences in their therapeutic activities? What do we make of the hypothesis that trastuzumab and pertuzumab simply mediate immune responses to HER2-positive tumors?

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AU Cancer Research Journal Club Slide 21