Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly

Nidhi Walia; Thais Pedrete; Fozieh Ahmadizadeh; Ekhtiar Rahman; Yashika Garg; Brian Washburn; Cheryl Pye; Fanny C. Liu; Peter Randolph; Kevin L. Weiss; Gergely Nagy; Christian Bleiholder; M. Elizabeth Stroupe

doi:10.1002/pro.70709

Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly

Nidhi Walia
, Thais Pedrete
, Fozieh Ahmadizadeh
, Ekhtiar Rahman
, Yashika Garg
, Brian Washburn
, Cheryl Pye
, Fanny C. Liu
, Peter Randolph
, Kevin L. Weiss
, Gergely Nagy
, Christian Bleiholder
, M. Elizabeth Stroupe

Research output: Contribution to journal › Article › peer-review

Abstract

How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology. NADPH-dependent assimilatory sulfite reductase (SiR) forms a heterododecamer built on an octameric flavoprotein (SiRFP) core, yet the molecular basis for this assembly has been unresolved because of its disordered N-terminus. Here, we use ion mobility mass spectrometry, small-angle neutron scattering, and mutagenesis to define the mechanism of SiRFP oligomerization. We show that SiRFP forms a discrete, stable octamer in solution. We also report that its N-terminal 52-residue segment is necessary and sufficient to mediate assembly, also mediating oligomerization when fused to a heterologous protein. Structure-guided mutagenesis identifies four residues (Gln22, Tyr39, Phe40, and Gln47) whose substitution disrupts the octamer, producing concentration-dependent lower-order species while retaining catalytic activity. These findings define the determinants of SiRFP assembly with broader implications for engineering homomeric protein complexes.

Original language	English
Article number	e70709
Journal	Protein Science
Volume	35
Issue number	8
DOIs	https://doi.org/10.1002/pro.70709
State	Published - Aug 2026

Funding

We thank Drs. Christopher Stroupe and Ratkim Roy for helpful discussions. A portion of this research used resources at Spallation Neutron Source, a Department of Energy office of Science User Facility operated by the Oak Ridge National Laboratory. The beam time was allocated to EQ-SANS on proposal number IPTS-30534. The Office of Biological and Environmental Research also supported work at the ORNL Center for Structural Molecular Biology. This research was supported in part by an appointment to the Oak Ridge National Laboratory Graduate Research at ORNL Program, sponsored by the U.S. Department of Energy and administered by the Oak Ridge Institute for Science and Education. Further support came from National Science Foundation grants MCB1856502 and CHE1904612 to M. Elizabeth Stroupe and CHE 2305173 to Christian Bleiholder and Fanny C. Liu and National Institutes of Health grant R01GM135682 to Christian Bleiholder. We thank Drs. Christopher Stroupe and Ratkim Roy for helpful discussions. A portion of this research used resources at Spallation Neutron Source, a Department of Energy office of Science User Facility operated by the Oak Ridge National Laboratory. The beam time was allocated to EQ‐SANS on proposal number IPTS‐30534. The Office of Biological and Environmental Research also supported work at the ORNL Center for Structural Molecular Biology. This research was supported in part by an appointment to the Oak Ridge National Laboratory Graduate Research at ORNL Program, sponsored by the U.S. Department of Energy and administered by the Oak Ridge Institute for Science and Education. Further support came from National Science Foundation grants MCB1856502 and CHE1904612 to M. Elizabeth Stroupe and CHE 2305173 to Christian Bleiholder and Fanny C. Liu and National Institutes of Health grant R01GM135682 to Christian Bleiholder.

Keywords

enzyme oligomerization
native ion mobility spectrometry/mass spectrometry
oxidoreductase
small-angle neutron scattering
sulfite reductase

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10.1002/pro.70709

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Walia, N., Pedrete, T., Ahmadizadeh, F., Rahman, E., Garg, Y., Washburn, B., Pye, C., Liu, F. C., Randolph, P., Weiss, K. L., Nagy, G., Bleiholder, C., & Stroupe, M. E. (2026). Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly. Protein Science, 35(8), Article e70709. https://doi.org/10.1002/pro.70709

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title = "Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly",

abstract = "How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology. NADPH-dependent assimilatory sulfite reductase (SiR) forms a heterododecamer built on an octameric flavoprotein (SiRFP) core, yet the molecular basis for this assembly has been unresolved because of its disordered N-terminus. Here, we use ion mobility mass spectrometry, small-angle neutron scattering, and mutagenesis to define the mechanism of SiRFP oligomerization. We show that SiRFP forms a discrete, stable octamer in solution. We also report that its N-terminal 52-residue segment is necessary and sufficient to mediate assembly, also mediating oligomerization when fused to a heterologous protein. Structure-guided mutagenesis identifies four residues (Gln22, Tyr39, Phe40, and Gln47) whose substitution disrupts the octamer, producing concentration-dependent lower-order species while retaining catalytic activity. These findings define the determinants of SiRFP assembly with broader implications for engineering homomeric protein complexes.",

keywords = "enzyme oligomerization, native ion mobility spectrometry/mass spectrometry, oxidoreductase, small-angle neutron scattering, sulfite reductase",

author = "Nidhi Walia and Thais Pedrete and Fozieh Ahmadizadeh and Ekhtiar Rahman and Yashika Garg and Brian Washburn and Cheryl Pye and Liu, \{Fanny C.\} and Peter Randolph and Weiss, \{Kevin L.\} and Gergely Nagy and Christian Bleiholder and Stroupe, \{M. Elizabeth\}",

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doi = "10.1002/pro.70709",

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journal = "Protein Science",

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Walia, N, Pedrete, T, Ahmadizadeh, F, Rahman, E, Garg, Y, Washburn, B, Pye, C, Liu, FC, Randolph, P, Weiss, KL , Nagy, G, Bleiholder, C & Stroupe, ME 2026, 'Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly', Protein Science, vol. 35, no. 8, e70709. https://doi.org/10.1002/pro.70709

TY - JOUR

T1 - Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly

AU - Walia, Nidhi

AU - Pedrete, Thais

AU - Ahmadizadeh, Fozieh

AU - Rahman, Ekhtiar

AU - Garg, Yashika

AU - Washburn, Brian

AU - Pye, Cheryl

AU - Liu, Fanny C.

AU - Randolph, Peter

AU - Weiss, Kevin L.

AU - Nagy, Gergely

AU - Bleiholder, Christian

AU - Stroupe, M. Elizabeth

PY - 2026/8

Y1 - 2026/8

N2 - How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology. NADPH-dependent assimilatory sulfite reductase (SiR) forms a heterododecamer built on an octameric flavoprotein (SiRFP) core, yet the molecular basis for this assembly has been unresolved because of its disordered N-terminus. Here, we use ion mobility mass spectrometry, small-angle neutron scattering, and mutagenesis to define the mechanism of SiRFP oligomerization. We show that SiRFP forms a discrete, stable octamer in solution. We also report that its N-terminal 52-residue segment is necessary and sufficient to mediate assembly, also mediating oligomerization when fused to a heterologous protein. Structure-guided mutagenesis identifies four residues (Gln22, Tyr39, Phe40, and Gln47) whose substitution disrupts the octamer, producing concentration-dependent lower-order species while retaining catalytic activity. These findings define the determinants of SiRFP assembly with broader implications for engineering homomeric protein complexes.

AB - How large, flexible enzymes assemble into defined oligomeric architectures remains a central question in biology. NADPH-dependent assimilatory sulfite reductase (SiR) forms a heterododecamer built on an octameric flavoprotein (SiRFP) core, yet the molecular basis for this assembly has been unresolved because of its disordered N-terminus. Here, we use ion mobility mass spectrometry, small-angle neutron scattering, and mutagenesis to define the mechanism of SiRFP oligomerization. We show that SiRFP forms a discrete, stable octamer in solution. We also report that its N-terminal 52-residue segment is necessary and sufficient to mediate assembly, also mediating oligomerization when fused to a heterologous protein. Structure-guided mutagenesis identifies four residues (Gln22, Tyr39, Phe40, and Gln47) whose substitution disrupts the octamer, producing concentration-dependent lower-order species while retaining catalytic activity. These findings define the determinants of SiRFP assembly with broader implications for engineering homomeric protein complexes.

KW - enzyme oligomerization

KW - native ion mobility spectrometry/mass spectrometry

KW - oxidoreductase

KW - small-angle neutron scattering

KW - sulfite reductase

UR - https://www.scopus.com/pages/publications/105044448182

U2 - 10.1002/pro.70709

DO - 10.1002/pro.70709

M3 - Article

AN - SCOPUS:105044448182

SN - 0961-8368

VL - 35

JO - Protein Science

JF - Protein Science

IS - 8

M1 - e70709

ER -

Critical amino acid residues in the N-terminal domain of NADPH-dependent assimilatory sulfite reductase flavoprotein mediate octameric assembly

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