Soumya De | Indian Institute of Technology Kharagpur (original) (raw)

Papers by Soumya De

Journal of Biomolecular NMR, 2011

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the pe... more The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, and apparent Michaelis constants, . By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13 C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide 13 C-1 H constant time HSQC spectra to determine , , , and for the catalytic domain of Pin1 acting on this AICD substrate. The onenzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

Peptidyl prolyl cis−trans isomerization acts as an effective molecular timer that plays significa... more Peptidyl prolyl cis−trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis−trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis−trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis−trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis−trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis−trans molecular switches and isomer-specific interactions involved in various biological processes.

Pathogenic bacteria have developed extraordinary strategies for invading host cells. The highly c... more Pathogenic bacteria have developed extraordinary strategies for invading host cells. The highly conserved type III secretion system (T3SS) provides a regulated conduit between the bacterial and host cytoplasm for delivery of a specific set of bacterial effector proteins that serve to disrupt host signaling and metabolism for the benefit of the bacterium. Remarkably, the inner diameter of the T3SS apparatus requires that effector proteins pass through in at least a partially unfolded form. AvrPto, an effector protein of the plant pathogen Pseudomonas syringae, adopts a helical bundle fold of low stability (G F3 U 2 kcal/mol at pH 7, 26.6 °C) and offers a model system for chaperone-independent secretion. P. syringae effector proteins encounter a pH gradient as they translocate from the bacterial cytoplasm (mildly acidic) into the host cell (neutral). Here, we demonstrate that AvrPto possesses a pH-sensitive folding switch controlled by conserved residue H87 that operates precisely in the pH range expected between the bacterial and host cyto-plasm environments. These results provide a mechanism for how a bacterial effector protein employs an intrinsic pH sensor to unfold for translocation via the T3SS and refold once in the host cytoplasm and provide fundamental insights for developing strategies for delivery of engineered therapeutic proteins to target tissues.

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the pe... more The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation , rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, k cis cat and apparent Michaelis constants, K App M. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13 C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phos-phopeptide 13 C– 1 H constant time HSQC spectra to determine k cis cat , k trans cat , K cis D , and K trans D for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [EÁtrans]/[EÁcis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPI-ase mutants to gain insights on the catalytic mechanism of this important enzyme.

Human cardiac myosin binding protein C (cMyBP-C), a thick filament protein found within the sarco... more Human cardiac myosin binding protein C (cMyBP-C), a thick filament protein found within the sarcomere of cardiac muscle, regulates muscle contraction and is essential for proper muscle function. Hypertrophic cardiomyopathy (HCM), a genetic disease affecting 1 in 500 people, is the major cause of death in young athletes. It is caused by genetic mutations within sarcomeric proteins. Forty-two percent of the HCM-related mutations are found in cMyBP-C. Here we present the nuclear magnetic resonance-derived structural ensembles of the wild-type cMyBP-C C3 domain and its HCM-related R502W mutant. The C3 domain adopts an immunoglobulin-like fold, and mutation of the exposed Arg502 to a tryptophan does not perturb its structure, dynamics, or stability. However, the R502W mutation does alter the predicted electrostatic properties of the C3 domain. We hypothesize that this mutation, and other HCM-linked mutations found within the same domain, may directly disrupt the interaction of cMyBP-C with other sarcomeric proteins.

DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~ 50-fold due to... more DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~ 50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus 5′ GGAA 3′ recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~ 5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing 5′ GGAA 3′ versus 5′ GGAT 3′. These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.

The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically bl... more The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge–dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity.

This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright ETV6 (or TEL), a transcriptional repressor belonging to the ETS family, is frequently involved in chromosomal translocations linked with human cancers. It displays a DNA-binding mode distinct from other ETS proteins due to the presence of a self-associating PNT domain. In this study, we used NMR spectroscopy to dissect the structural and dynamic bases for the autoinhibition of ETV6 DNA binding by sequences C-terminal to its ETS domain. The C-terminal inhibitory domain (CID) contains two helices, H4 and H5, which sterically block the DNA-binding interface of the ETS domain. Importantly, these appended helices are only marginally stable as revealed by amide hydrogen exchange and 15 N relaxation measurements. The CID is thus poised to undergo a facile conformational change as required for DNA binding. The CID also dampens millisecond timescale motions of the ETS domain hypothesized to be critical for the recognition of specific ETS target sequences. This work illustrates the use of appended sequences on conserved structural domains to generate biological diversity and complements previous studies of the allosteric mechanism of ETS1 autoinhibition to reveal both common and divergent features underlying the regulation of DNA binding by ETS transcription factors.

SUMMARY The type III secretion system (T3SS) is a large macro-molecular assembly found at the sur... more SUMMARY The type III secretion system (T3SS) is a large macro-molecular assembly found at the surface of many pathogenic Gram-negative bacteria. Its role is to inject toxic ''effector'' proteins into the cells of infected organisms. The molecular details of the assembly of this large, multimembrane-spanning complex remain poorly understood. Here, we report structural, biochemical, and functional analyses of PrgK, an inner-membrane component of the proto-typical Salmonella typhimurium T3SS. We have obtained the atomic structures of the two ring building globular domains and show that the C-terminal transmembrane helix is not essential for assembly and secretion. We also demonstrate that structural rearrangement of the two PrgK globular domains, driven by an interconnecting linker region, may promote oligomerization into ring structures. Finally, we used electron microscopy-guided symmetry modeling to propose a structural model for the intimately associated PrgH-PrgK ring interaction within the assembled basal body.

Journal of Biomolecular NMR, 2011

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the pe... more The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation, rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, and apparent Michaelis constants, . By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13 C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phosphopeptide 13 C-1 H constant time HSQC spectra to determine , , , and for the catalytic domain of Pin1 acting on this AICD substrate. The onenzyme equilibrium value of [E·trans]/[E·cis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPIase mutants to gain insights on the catalytic mechanism of this important enzyme.

Peptidyl prolyl cis−trans isomerization acts as an effective molecular timer that plays significa... more Peptidyl prolyl cis−trans isomerization acts as an effective molecular timer that plays significant roles in biological and pathological processes. Enzymes such as Pin1 catalyze cis−trans isomerization, accelerating the otherwise slow isomerization rate into time scales relevant for cellular signaling. Here we have combined NMR line shape analysis, fluorescence spectroscopy, and isothermal titration calorimetry to determine the kinetic and thermodynamic parameters describing the trans-specific interaction between the binding domain of Pin1 (WW domain) and a key cis−trans molecular switch in the amyloid precursor protein cytoplasmic tail. A three-state model, in which the cis−trans isomerization equilibrium is coupled to the binding equilibrium through the trans isomer, was found to fit the data well. The trans isomer binds the WW domain with ∼22 μM affinity via very fast association (approaching the diffusion limit) and dissociation rates. The common structural and electrostatic characteristics of Pin1 substrates, which contain a phosphorylated serine/threonine-proline motif, suggest that very rapid binding kinetics are a general feature of Pin1 interactions with other substrates. The fast binding kinetics of the WW domain allows rapid response of Pin1 to the dynamic events of phosphorylation and dephosphorylation in the cell that alter the relative populations of diverse Pin1 substrates. Furthermore, our results also highlight the vastly different rates at which slow uncatalyzed cis−trans isomerization and fast isomer-specific binding events occur. These results, along with the experimental methods presented herein, should guide future experiments aimed at the thermodynamic and kinetic characterization of cis−trans molecular switches and isomer-specific interactions involved in various biological processes.

Pathogenic bacteria have developed extraordinary strategies for invading host cells. The highly c... more Pathogenic bacteria have developed extraordinary strategies for invading host cells. The highly conserved type III secretion system (T3SS) provides a regulated conduit between the bacterial and host cytoplasm for delivery of a specific set of bacterial effector proteins that serve to disrupt host signaling and metabolism for the benefit of the bacterium. Remarkably, the inner diameter of the T3SS apparatus requires that effector proteins pass through in at least a partially unfolded form. AvrPto, an effector protein of the plant pathogen Pseudomonas syringae, adopts a helical bundle fold of low stability (G F3 U 2 kcal/mol at pH 7, 26.6 °C) and offers a model system for chaperone-independent secretion. P. syringae effector proteins encounter a pH gradient as they translocate from the bacterial cytoplasm (mildly acidic) into the host cell (neutral). Here, we demonstrate that AvrPto possesses a pH-sensitive folding switch controlled by conserved residue H87 that operates precisely in the pH range expected between the bacterial and host cyto-plasm environments. These results provide a mechanism for how a bacterial effector protein employs an intrinsic pH sensor to unfold for translocation via the T3SS and refold once in the host cytoplasm and provide fundamental insights for developing strategies for delivery of engineered therapeutic proteins to target tissues.

The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the pe... more The phosphorylation-specific peptidyl-prolyl isomerase Pin1 catalyzes the isomerization of the peptide bond preceding a proline residue between cis and trans isomers. To best understand the mechanisms of Pin1 regulation , rigorous enzymatic assays of isomerization are required. However, most measures of isomerase activity require significant constraints on substrate sequence and only yield rate constants for the cis isomer, k cis cat and apparent Michaelis constants, K App M. By contrast, NMR lineshape analysis is a powerful tool for determining microscopic rates and populations of each state in a complex binding scheme. The isolated catalytic domain of Pin1 was employed as a first step towards elucidating the reaction scheme of the full-length enzyme. A 24-residue phosphopeptide derived from the amyloid precurser protein intracellular domain (AICD) phosphorylated at Thr668 served as a biologically-relevant Pin1 substrate. Specific 13 C labeling at the Pin1-targeted proline residue provided multiple reporters sensitive to individual isomer binding and on-enzyme catalysis. We have performed titration experiments and employed lineshape analysis of phos-phopeptide 13 C– 1 H constant time HSQC spectra to determine k cis cat , k trans cat , K cis D , and K trans D for the catalytic domain of Pin1 acting on this AICD substrate. The on-enzyme equilibrium value of [EÁtrans]/[EÁcis] = 3.9 suggests that the catalytic domain of Pin1 is optimized to operate on this substrate near equilibrium in the cellular context. This highlights the power of lineshape analysis for determining the microscopic parameters of enzyme catalysis, and demonstrates the feasibility of future studies of Pin1-PPI-ase mutants to gain insights on the catalytic mechanism of this important enzyme.

Human cardiac myosin binding protein C (cMyBP-C), a thick filament protein found within the sarco... more Human cardiac myosin binding protein C (cMyBP-C), a thick filament protein found within the sarcomere of cardiac muscle, regulates muscle contraction and is essential for proper muscle function. Hypertrophic cardiomyopathy (HCM), a genetic disease affecting 1 in 500 people, is the major cause of death in young athletes. It is caused by genetic mutations within sarcomeric proteins. Forty-two percent of the HCM-related mutations are found in cMyBP-C. Here we present the nuclear magnetic resonance-derived structural ensembles of the wild-type cMyBP-C C3 domain and its HCM-related R502W mutant. The C3 domain adopts an immunoglobulin-like fold, and mutation of the exposed Arg502 to a tryptophan does not perturb its structure, dynamics, or stability. However, the R502W mutation does alter the predicted electrostatic properties of the C3 domain. We hypothesize that this mutation, and other HCM-linked mutations found within the same domain, may directly disrupt the interaction of cMyBP-C with other sarcomeric proteins.

DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~ 50-fold due to... more DNA binding by the ETS transcriptional repressor ETV6 (or TEL) is auto-inhibited ~ 50-fold due to an α-helix that sterically blocks its ETS domain binding interface. Using NMR spectroscopy, we demonstrate that this marginally stable helix is unfolded, and not displaced to a non-inhibitory position, when ETV6 is bound to DNA containing a consensus 5′ GGAA 3′ recognition site. Although significantly lower in affinity, binding to non-specific DNA is auto-inhibited ~ 5-fold and is also accompanied by helix unfolding. Based on NMR chemical shift perturbations, both specific and non-specific DNA are bound via the same canonical ETS domain interface. However, spectral perturbations are smaller for the non-specific complex, suggesting weaker and less well-defined interactions than in the specific complex. In parallel, the crystal structure of ETV6 bound to a specific DNA duplex was determined. The structure of this complex reveals that a non-conserved histidine residue in the ETS domain recognition helix helps establish the specificity of ETV6 for DNA-binding sites containing 5′ GGAA 3′ versus 5′ GGAT 3′. These studies provide a unified steric mechanism for attenuating ETV6 binding to both specific and non-specific DNA and expand the repertoire of characterized auto-inhibitory strategies utilized to regulate ETS factors.

The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically bl... more The ETS transcriptional repressor ETV6 (or TEL) is autoinhibited by an α-helix that sterically blocks its DNA-binding ETS domain. The inhibitory helix is marginally stable and unfolds when ETV6 binds to either specific or non-specific DNA. Using NMR spectroscopy, we show that folding of the inhibitory helix requires a buried charge–dipole interaction with helix H1 of the ETS domain. This interaction also contributes directly to autoinhibition by precluding a highly conserved dipole-enhanced hydrogen bond between the phosphodiester backbone of bound DNA and the N terminus of helix H1. To probe further the thermodynamic basis of autoinhibition, ETV6 variants were generated with amino acid substitutions introduced along the solvent exposed surface of the inhibitory helix. These changes were designed to increase the intrinsic helical propensity of the inhibitory helix without perturbing its packing interactions with the ETS domain. NMR-monitored amide hydrogen exchange measurements confirmed that the stability of the folded inhibitory helix increases progressively with added helix-promoting substitutions. This also results in progressively reinforced autoinhibition and decreased DNA-binding affinity. Surprisingly, locking the inhibitory helix onto the ETS domain by a disulfide bridge severely impairs, but does not abolish DNA binding. Weak interactions still occur via an interface displaced from the canonical ETS domain DNA-binding surface. Collectively, these studies establish a direct thermodynamic linkage between inhibitory helix stability and ETV6 autoinhibition, and demonstrate that helix unfolding does not strictly precede DNA binding. Modulating inhibitory helix stability provides a potential route for the in vivo regulation of ETV6 activity.

This article appeared in a journal published by Elsevier. The attached copy is furnished to the a... more This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier's archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright ETV6 (or TEL), a transcriptional repressor belonging to the ETS family, is frequently involved in chromosomal translocations linked with human cancers. It displays a DNA-binding mode distinct from other ETS proteins due to the presence of a self-associating PNT domain. In this study, we used NMR spectroscopy to dissect the structural and dynamic bases for the autoinhibition of ETV6 DNA binding by sequences C-terminal to its ETS domain. The C-terminal inhibitory domain (CID) contains two helices, H4 and H5, which sterically block the DNA-binding interface of the ETS domain. Importantly, these appended helices are only marginally stable as revealed by amide hydrogen exchange and 15 N relaxation measurements. The CID is thus poised to undergo a facile conformational change as required for DNA binding. The CID also dampens millisecond timescale motions of the ETS domain hypothesized to be critical for the recognition of specific ETS target sequences. This work illustrates the use of appended sequences on conserved structural domains to generate biological diversity and complements previous studies of the allosteric mechanism of ETS1 autoinhibition to reveal both common and divergent features underlying the regulation of DNA binding by ETS transcription factors.

SUMMARY The type III secretion system (T3SS) is a large macro-molecular assembly found at the sur... more SUMMARY The type III secretion system (T3SS) is a large macro-molecular assembly found at the surface of many pathogenic Gram-negative bacteria. Its role is to inject toxic ''effector'' proteins into the cells of infected organisms. The molecular details of the assembly of this large, multimembrane-spanning complex remain poorly understood. Here, we report structural, biochemical, and functional analyses of PrgK, an inner-membrane component of the proto-typical Salmonella typhimurium T3SS. We have obtained the atomic structures of the two ring building globular domains and show that the C-terminal transmembrane helix is not essential for assembly and secretion. We also demonstrate that structural rearrangement of the two PrgK globular domains, driven by an interconnecting linker region, may promote oligomerization into ring structures. Finally, we used electron microscopy-guided symmetry modeling to propose a structural model for the intimately associated PrgH-PrgK ring interaction within the assembled basal body.