Keith Inman - Academia.edu (original) (raw)

Papers by Keith Inman

Research paper thumbnail of Refinement of the solution structure and dynamic properties of Ca 2+ -bound rat S100B

Journal of Biomolecular Nmr, 2008

Biological contextThe small (~10.5 kDa) acidic Ca2+-binding protein S100B belongs to the S100 pro... more Biological contextThe small (~10.5 kDa) acidic Ca2+-binding protein S100B belongs to the S100 protein family, a group of over 20 members that share significant sequence homology and undergo a large conformational change upon the addition of calcium. S100 proteins have no intrinsic enzymatic activity; instead, a Ca2+-dependent conformational change enables them to bind and modulate the activity of a diverse pool of intracellular target proteins. For S100B, these include cytoskeletal and filament associated proteins (e.g. tubulin, GFAP, tau, desmin, vimentin, CapZ, calponin, calpactin I, and caldesmon), other Ca2+-binding proteins (annexins II, V, VI, S100A1, S100A6, S100A11, and neurocalcin-δ), membrane associated proteins (neuromodulin, neurogranin, MARCKS, giant protein AHNAK, and IQGAP1), transcription factors and their regulators (e.g. p53, hdm4, and hdm2), and several enzymes (e.g. aldolase, phosphoglucomutase, photoreceptor guanyl cyclases, Ndr kinase, and protein kinase C) (revie

Research paper thumbnail of Backbone Dynamics of the Calcium-Signaling Protein apo-S100B as Determined by 15 N NMR Relaxation

Biochemistry, 2001

Backbone dynamics of homodimeric apo-S100B were studied by 15 N nuclear magnetic resonance relaxa... more Backbone dynamics of homodimeric apo-S100B were studied by 15 N nuclear magnetic resonance relaxation at 9.4 and 14.1 T. Longitudinal relaxation (T 1 ), transverse relaxation (T 2 ), and the 15 N-{ 1 H} NOE were measured for 80 of 91 backbone amide groups. Internal motional parameters were determined from the relaxation data using the model-free formalism while accounting for diffusion anisotropy. Rotational diffusion of the symmetric homodimer has moderate but statistically significant prolate axial anisotropy (D | /D ⊥ ) 1.15 ( 0.02), a global correlation time of τ m ) 7.80 ( 0.03 ns, and a unique axis in the plane normal to the molecular symmetry axis. Of 29 residues at the dimer interface (helices 1 and 4), only one has measurable internal motion (Q71), and the order parameters of the remaining 28 were the highest in the protein (S 2 ) 0.80 to 0.91). Order parameters in the typical EF hand calciumbinding loop (S 2 ) 0.73 to 0.87) were slightly lower than in the pseudo-EF hand (S 2 ) 0.75 to 0.89), and effective internal correlation times, τ e , distinct from global tumbling, were detected in the calcium-binding loops. Helix 3, which undergoes a large, calcium-induced conformational change necessary for targetprotein binding, does not show evidence of interchanging between the apo and Ca 2+ -bound orientations in the absence of calcium but has rapid motion in several residues throughout the helix (S 2 ) 0.78 to 0.88; 10 e τ e e 30 ps). The lowest order parameters were found in the C-terminal tail (S 2 ) 0.62 to 0.83). Large values for chemical exchange also occur in this loop and in regions nearby in space to the highly mobile C-terminal loop, consistent with exchange broadening effects observed. ; S100 , subunit of dimeric S100B; S100B( ), dimeric S100B with noncovalent interactions at the dimer interface; sNTnC, N-terminal domain of skeletal troponin C; PKC, protein kinase C; RMSD, root-mean-square difference; NOE, nuclear Overhauser effect; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence; TPPI time-proportional phase incrementation; ψ-EF, pseudo-EF hand.

Research paper thumbnail of The Major Pilin Subunit of the AAF/II Fimbriae from Enteroaggregative Escherichia coli Mediates Binding to Extracellular Matrix Proteins

Research paper thumbnail of Three-Dimensional Solution Structure of the Calcium-Signaling Protein Apo-S100A1 As Determined by NMR

Biochemistry, 2002

S100A1, a member of the S100 protein family, is an EF-hand containing Ca 2+ -binding protein (93 ... more S100A1, a member of the S100 protein family, is an EF-hand containing Ca 2+ -binding protein (93 residues per subunit) with noncovalent interactions at its dimer interface. Each subunit of S100A1 has four R-helices and a small antiparallel -sheet consistent with two helix-loop-helix calcium-binding domains [Baldiserri et al. (1999) J. Biomol. NMR 14, 87-88]. In this study, the three-dimensional structure of reduced apo-S100A1 was determined by NMR spectroscopy using a total of 2220 NOE distance constraints, 258 dihedral angle constraints, and 168 backbone hydrogen bond constraints derived from a series of 2D, 3D, and 4D NMR experiments. The final structure was found to be globular and compact with the four helices in each subunit aligning to form a unicornate-type four-helix bundle. Intermolecular NOE correlations were observed between residues in helices 1 and 4 from one subunit to residues in helices 1′ and 4′ of the other subunit, respectively, consistent with the antiparallel alignment of the two subunits to form a symmetric X-type four-helix bundle as found for other members of the S100 protein family. Because of the similarity of the S100A1 dimer interface to that found for S100B, it was possible to calculate a model of the S100A1/B heterodimer. This model is consistent with a number of NMR chemical shift changes observed when S100A1 is titrated into a sample of 15 N-labeled S100B. Helix 3 (and 3′) of S100A1 was found to have an interhelical angle of -150°with helix 4 (and 4′) in the apo state. This crossing angle is quite different (>50°) from that typically found in other EF-hand containing proteins such as apocalmodulin and apotroponin C but more similar to apo-S100B, which has an interhelical angle of -166°. As with S100B, it is likely that the second EF-hand of apo-S100A1 reorients dramatically upon the addition of Ca 2+ , which can explain the Ca 2+ dependence that S100A1 has for binding several of its biological targets. ; S100A1, homodimeric S100A1-(RR) with noncovalent interactions at the dimer interface; S100R, subunit of dimeric S100A1; S100 , subunit of dimeric S100B; S100B-( ) or S100B, dimeric S100B with noncovalent interactions at the dimer interface; S100A1/B, heterodimer that has a single S100A1 R-subunit and a single S100B -subunit held together noncovalently; rmsd, root mean square difference; NOE, nuclear Overhauser effect; NMR, nuclear magnetic resonance; HSQC, heteronuclear singlequantum coherence; TPPI, time-proportional phase incrementation; 3D, three dimensional.

Research paper thumbnail of Solution NMR Structure of S100B Bound to the High-affinity Target Peptide TRTK-12

Journal of Molecular Biology, 2002

The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRT... more The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (alpha1 or alpha2 subunit, residues 265-276: TRTKIDWNKILS). This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL). As with other S100B target proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding. The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B-TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding. However, helical TRTK-12 in the S100B-peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100-peptide complexes. The three-dimensional structure of the S100B-TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B-peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity. A comparison of the S100B-TRTK peptide structure to the structures of apo- and Ca(2+)-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca(2+)-bound S100B as necessary to bind the TRTK-12 peptide.

Research paper thumbnail of Solution structure of the novel dispersin protein of enteroaggregative Escherichia coli

Molecular Microbiology, 2007

Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infan... more Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked-brick pattern of adherence to epithelial cells. Adherence is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid. We recently described a highly prevalent pAA plasmid-borne gene, aap, which encodes a protein (nicknamed dispersin) that is secreted to the bacterial cell surface. Dispersin-null mutants display a unique hyper-aggregating phenotype, accompanied by collapse of AAF pili onto the bacterial cell surface. To study the mechanism of this effect, we solved the structure of dispersin from EAEC strain 042 using solution NMR, revealing a stable beta-sandwich with a conserved net positive surface charge of +3 to +4 among 23 dispersin alleles. Experimental data suggest that dispersin binds non-covalently to lipopolysaccharide on the surface of the bacterium. We also show that the AAF organelles contribute positive charge to the bacterial surface, suggesting that dispersin's role in fimbrial function is to overcome electrostatic attraction between AAF and the bacterial surface.

Research paper thumbnail of A Bayesian Statistical Method for the Detection and Quantification of Rotational Diffusion Anisotropy from NMR Relaxation Data

Journal of Magnetic Resonance, 2000

It has recently become more widely appreciated that the presence of rotational diffusional anisot... more It has recently become more widely appreciated that the presence of rotational diffusional anisotropy in proteins and other macromolecules can have a significant affect on the interpretation of NMR relaxation data in terms of molecular motion. In this paper, we show how commonly used NMR relaxation data (R 1 , R 2 , and NOE) obtained at two spectrometer frequencies can be analyzed using a Bayesian statistical approach to reliably detect and quantify the degree of rotational diffusion anisotropy. Our approach differs from previous methods in that it does not make assumptions concerning the internal motions experienced by the residues which are used to quantify the diffusion anisotropy, but rather averages the results over all internal motions consistent with the data. We demonstrate our method using synthetic data corresponding to isotropic, axially symmetric anisotropic, and fully asymmetric anisotropic rotational diffusion, as well as experimental NMR data. We compare the Bayesian statistical approach with a widely used method for extracting tumbling parameters using both synthetic and experimental data. While it can be difficult to separate the effects of chemical exchange from rotational anisotropy using this "standard" method, these effects are readily separated using Bayesian statistics. In addition, we find that the Bayesian statistical approach requires considerably less CPU time than an equivalent standard analysis.

Research paper thumbnail of Complete 1 H, 13 C and 15 N NMR assignments for donor-strand complemented AafA, the major pilin of aggregative adherence fimbriae (AAF/II) from enteroaggregative E. coli

Biomolecular Nmr Assignments, 2011

Aggregative adherence fimbriae (AAF) are the primary adhesive factors of enteroaggregative Escher... more Aggregative adherence fimbriae (AAF) are the primary adhesive factors of enteroaggregative Escherichia coli (EAEC) and are required for intestinal colonization. They mediate binding to extracellular matrix proteins of the enteric mucosa and display proinflammatory effects on epithelial cells in vitro. Among the simplest of bacterial fimbriae, these passive hairlike appendages are composed primarily of a single 16-kDa structural and adhesive subunit, AafA. Oligomerization occurs by incorporating the N-terminal strand of each AafA subunit into an otherwise incomplete β-sheet of an adjacent AafA subunit. We have engineered a highly soluble AafA monomer by positioning the N-terminal “donor strand” at the C-terminus, following a turn and short linker that were introduced to allow access of the donor strand to the recipient cleft of the same subunit. The resulting “donor-strand complemented” AafA subunit, or AafA-dsc folds autonomously, is monodisperse in solution, and yields high quality NMR spectral data. Here, we report the 1H, 13C, and 15N chemical shift assignments for AafA-dsc.

Research paper thumbnail of Refinement of the solution structure and dynamic properties of Ca 2+ -bound rat S100B

Journal of Biomolecular Nmr, 2008

Biological contextThe small (~10.5 kDa) acidic Ca2+-binding protein S100B belongs to the S100 pro... more Biological contextThe small (~10.5 kDa) acidic Ca2+-binding protein S100B belongs to the S100 protein family, a group of over 20 members that share significant sequence homology and undergo a large conformational change upon the addition of calcium. S100 proteins have no intrinsic enzymatic activity; instead, a Ca2+-dependent conformational change enables them to bind and modulate the activity of a diverse pool of intracellular target proteins. For S100B, these include cytoskeletal and filament associated proteins (e.g. tubulin, GFAP, tau, desmin, vimentin, CapZ, calponin, calpactin I, and caldesmon), other Ca2+-binding proteins (annexins II, V, VI, S100A1, S100A6, S100A11, and neurocalcin-δ), membrane associated proteins (neuromodulin, neurogranin, MARCKS, giant protein AHNAK, and IQGAP1), transcription factors and their regulators (e.g. p53, hdm4, and hdm2), and several enzymes (e.g. aldolase, phosphoglucomutase, photoreceptor guanyl cyclases, Ndr kinase, and protein kinase C) (revie

Research paper thumbnail of Backbone Dynamics of the Calcium-Signaling Protein apo-S100B as Determined by 15 N NMR Relaxation

Biochemistry, 2001

Backbone dynamics of homodimeric apo-S100B were studied by 15 N nuclear magnetic resonance relaxa... more Backbone dynamics of homodimeric apo-S100B were studied by 15 N nuclear magnetic resonance relaxation at 9.4 and 14.1 T. Longitudinal relaxation (T 1 ), transverse relaxation (T 2 ), and the 15 N-{ 1 H} NOE were measured for 80 of 91 backbone amide groups. Internal motional parameters were determined from the relaxation data using the model-free formalism while accounting for diffusion anisotropy. Rotational diffusion of the symmetric homodimer has moderate but statistically significant prolate axial anisotropy (D | /D ⊥ ) 1.15 ( 0.02), a global correlation time of τ m ) 7.80 ( 0.03 ns, and a unique axis in the plane normal to the molecular symmetry axis. Of 29 residues at the dimer interface (helices 1 and 4), only one has measurable internal motion (Q71), and the order parameters of the remaining 28 were the highest in the protein (S 2 ) 0.80 to 0.91). Order parameters in the typical EF hand calciumbinding loop (S 2 ) 0.73 to 0.87) were slightly lower than in the pseudo-EF hand (S 2 ) 0.75 to 0.89), and effective internal correlation times, τ e , distinct from global tumbling, were detected in the calcium-binding loops. Helix 3, which undergoes a large, calcium-induced conformational change necessary for targetprotein binding, does not show evidence of interchanging between the apo and Ca 2+ -bound orientations in the absence of calcium but has rapid motion in several residues throughout the helix (S 2 ) 0.78 to 0.88; 10 e τ e e 30 ps). The lowest order parameters were found in the C-terminal tail (S 2 ) 0.62 to 0.83). Large values for chemical exchange also occur in this loop and in regions nearby in space to the highly mobile C-terminal loop, consistent with exchange broadening effects observed. ; S100 , subunit of dimeric S100B; S100B( ), dimeric S100B with noncovalent interactions at the dimer interface; sNTnC, N-terminal domain of skeletal troponin C; PKC, protein kinase C; RMSD, root-mean-square difference; NOE, nuclear Overhauser effect; NMR, nuclear magnetic resonance; HSQC, heteronuclear single quantum coherence; TPPI time-proportional phase incrementation; ψ-EF, pseudo-EF hand.

Research paper thumbnail of The Major Pilin Subunit of the AAF/II Fimbriae from Enteroaggregative Escherichia coli Mediates Binding to Extracellular Matrix Proteins

Research paper thumbnail of Three-Dimensional Solution Structure of the Calcium-Signaling Protein Apo-S100A1 As Determined by NMR

Biochemistry, 2002

S100A1, a member of the S100 protein family, is an EF-hand containing Ca 2+ -binding protein (93 ... more S100A1, a member of the S100 protein family, is an EF-hand containing Ca 2+ -binding protein (93 residues per subunit) with noncovalent interactions at its dimer interface. Each subunit of S100A1 has four R-helices and a small antiparallel -sheet consistent with two helix-loop-helix calcium-binding domains [Baldiserri et al. (1999) J. Biomol. NMR 14, 87-88]. In this study, the three-dimensional structure of reduced apo-S100A1 was determined by NMR spectroscopy using a total of 2220 NOE distance constraints, 258 dihedral angle constraints, and 168 backbone hydrogen bond constraints derived from a series of 2D, 3D, and 4D NMR experiments. The final structure was found to be globular and compact with the four helices in each subunit aligning to form a unicornate-type four-helix bundle. Intermolecular NOE correlations were observed between residues in helices 1 and 4 from one subunit to residues in helices 1′ and 4′ of the other subunit, respectively, consistent with the antiparallel alignment of the two subunits to form a symmetric X-type four-helix bundle as found for other members of the S100 protein family. Because of the similarity of the S100A1 dimer interface to that found for S100B, it was possible to calculate a model of the S100A1/B heterodimer. This model is consistent with a number of NMR chemical shift changes observed when S100A1 is titrated into a sample of 15 N-labeled S100B. Helix 3 (and 3′) of S100A1 was found to have an interhelical angle of -150°with helix 4 (and 4′) in the apo state. This crossing angle is quite different (>50°) from that typically found in other EF-hand containing proteins such as apocalmodulin and apotroponin C but more similar to apo-S100B, which has an interhelical angle of -166°. As with S100B, it is likely that the second EF-hand of apo-S100A1 reorients dramatically upon the addition of Ca 2+ , which can explain the Ca 2+ dependence that S100A1 has for binding several of its biological targets. ; S100A1, homodimeric S100A1-(RR) with noncovalent interactions at the dimer interface; S100R, subunit of dimeric S100A1; S100 , subunit of dimeric S100B; S100B-( ) or S100B, dimeric S100B with noncovalent interactions at the dimer interface; S100A1/B, heterodimer that has a single S100A1 R-subunit and a single S100B -subunit held together noncovalently; rmsd, root mean square difference; NOE, nuclear Overhauser effect; NMR, nuclear magnetic resonance; HSQC, heteronuclear singlequantum coherence; TPPI, time-proportional phase incrementation; 3D, three dimensional.

Research paper thumbnail of Solution NMR Structure of S100B Bound to the High-affinity Target Peptide TRTK-12

Journal of Molecular Biology, 2002

The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRT... more The solution NMR structure is reported for Ca(2+)-loaded S100B bound to a 12-residue peptide, TRTK-12, from the actin capping protein CapZ (alpha1 or alpha2 subunit, residues 265-276: TRTKIDWNKILS). This peptide was discovered by Dimlich and co-workers by screening a bacteriophage random peptide display library, and it matches exactly the consensus S100B binding sequence ((K/R)(L/I)XWXXIL). As with other S100B target proteins, a calcium-dependent conformational change in S100B is required for TRTK-12 binding. The TRTK-12 peptide is an amphipathic helix (residues W7 to S12) in the S100B-TRTK complex, and helix 4 of S100B is extended by three or four residues upon peptide binding. However, helical TRTK-12 in the S100B-peptide complex is uniquely oriented when compared to the three-dimensional structures of other S100-peptide complexes. The three-dimensional structure of the S100B-TRTK peptide complex illustrates that residues in the S100B binding consensus sequence (K4, I5, W7, I10, L11) are all involved in the S100B-peptide interface, which can explain its orientation in the S100B binding pocket and its relatively high binding affinity. A comparison of the S100B-TRTK peptide structure to the structures of apo- and Ca(2+)-bound S100B illustrates that the binding site of TRTK-12 is buried in apo-S100B, but is exposed in Ca(2+)-bound S100B as necessary to bind the TRTK-12 peptide.

Research paper thumbnail of Solution structure of the novel dispersin protein of enteroaggregative Escherichia coli

Molecular Microbiology, 2007

Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infan... more Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked-brick pattern of adherence to epithelial cells. Adherence is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid. We recently described a highly prevalent pAA plasmid-borne gene, aap, which encodes a protein (nicknamed dispersin) that is secreted to the bacterial cell surface. Dispersin-null mutants display a unique hyper-aggregating phenotype, accompanied by collapse of AAF pili onto the bacterial cell surface. To study the mechanism of this effect, we solved the structure of dispersin from EAEC strain 042 using solution NMR, revealing a stable beta-sandwich with a conserved net positive surface charge of +3 to +4 among 23 dispersin alleles. Experimental data suggest that dispersin binds non-covalently to lipopolysaccharide on the surface of the bacterium. We also show that the AAF organelles contribute positive charge to the bacterial surface, suggesting that dispersin's role in fimbrial function is to overcome electrostatic attraction between AAF and the bacterial surface.

Research paper thumbnail of A Bayesian Statistical Method for the Detection and Quantification of Rotational Diffusion Anisotropy from NMR Relaxation Data

Journal of Magnetic Resonance, 2000

It has recently become more widely appreciated that the presence of rotational diffusional anisot... more It has recently become more widely appreciated that the presence of rotational diffusional anisotropy in proteins and other macromolecules can have a significant affect on the interpretation of NMR relaxation data in terms of molecular motion. In this paper, we show how commonly used NMR relaxation data (R 1 , R 2 , and NOE) obtained at two spectrometer frequencies can be analyzed using a Bayesian statistical approach to reliably detect and quantify the degree of rotational diffusion anisotropy. Our approach differs from previous methods in that it does not make assumptions concerning the internal motions experienced by the residues which are used to quantify the diffusion anisotropy, but rather averages the results over all internal motions consistent with the data. We demonstrate our method using synthetic data corresponding to isotropic, axially symmetric anisotropic, and fully asymmetric anisotropic rotational diffusion, as well as experimental NMR data. We compare the Bayesian statistical approach with a widely used method for extracting tumbling parameters using both synthetic and experimental data. While it can be difficult to separate the effects of chemical exchange from rotational anisotropy using this "standard" method, these effects are readily separated using Bayesian statistics. In addition, we find that the Bayesian statistical approach requires considerably less CPU time than an equivalent standard analysis.

Research paper thumbnail of Complete 1 H, 13 C and 15 N NMR assignments for donor-strand complemented AafA, the major pilin of aggregative adherence fimbriae (AAF/II) from enteroaggregative E. coli

Biomolecular Nmr Assignments, 2011

Aggregative adherence fimbriae (AAF) are the primary adhesive factors of enteroaggregative Escher... more Aggregative adherence fimbriae (AAF) are the primary adhesive factors of enteroaggregative Escherichia coli (EAEC) and are required for intestinal colonization. They mediate binding to extracellular matrix proteins of the enteric mucosa and display proinflammatory effects on epithelial cells in vitro. Among the simplest of bacterial fimbriae, these passive hairlike appendages are composed primarily of a single 16-kDa structural and adhesive subunit, AafA. Oligomerization occurs by incorporating the N-terminal strand of each AafA subunit into an otherwise incomplete β-sheet of an adjacent AafA subunit. We have engineered a highly soluble AafA monomer by positioning the N-terminal “donor strand” at the C-terminus, following a turn and short linker that were introduced to allow access of the donor strand to the recipient cleft of the same subunit. The resulting “donor-strand complemented” AafA subunit, or AafA-dsc folds autonomously, is monodisperse in solution, and yields high quality NMR spectral data. Here, we report the 1H, 13C, and 15N chemical shift assignments for AafA-dsc.