Therapeutic proteins contain a large number of post-translational modifications, some of which could potentially impact their safety or efficacy. In one of these changes, pyroglutamate can form on ...the N terminus of the polypeptide chain. Both glutamine and glutamate at the N termini of recombinant monoclonal antibodies can cyclize spontaneously to pyroglutamate (pE) in vitro. Glutamate conversion to pyroglutamate occurs more slowly than from glutamine but has been observed under near physiological conditions. Here we investigated to what extent human IgG2 N-terminal glutamate converts to pE in vivo. Pyroglutamate levels increased over time after injection into humans, with the rate of formation differing between polypeptide chains. These changes were replicated for the same antibodies in vitro under physiological pH and temperature conditions, indicating that the changes observed in vivo were due to chemical conversion not differential clearance. Differences in the conversion rates between the light chain and heavy chain on an antibody were eliminated by denaturing the protein, revealing that structural elements affect pE formation rates. By enzymatically releasing pE from endogenous antibodies isolated from human serum, we could estimate the naturally occurring levels of this post-translational modification. Together, these techniques and results can be used to predict the exposure of pE for therapeutic antibodies and to guide criticality assessments for this attribute.
The mechanosensitive channel of small conductance (MscS) responds both to stretching of the cell membrane and to membrane depolarization. The crystal structure at 3.9 angstroms resolution ...demonstrates that Escherichia coli MscS folds as a membrane-spanning heptamer with a large cytoplasmic region. Each subunit contains three transmembrane helices (TM1, -2, and -3), with the TM3 helices lining the pore, while TM1 and TM2, with membrane-embedded arginines, are likely candidates for the tension and voltage sensors. The transmembrane pore, apparently captured in an open state, connects to a large chamber, formed within the cytoplasmic region, that connects to the cytoplasm through openings that may function as molecular filters. Although MscS is likely to be structurally distinct from other ion channels, similarities in gating mechanisms suggest common structural elements.
Site-directed cysteine and disulfide chemistry is broadly useful in the analysis of protein structure and dynamics, and applications of this chemistry to the bacterial chemotaxis pathway have ...illustrated the kinds of information that can be generated. Notably, in many cases, cysteine and disulfide chemistry can be carried out in the native environment of the protein whether it be aqueous solution, a lipid bilayer, or a multiprotein complex. Moreover, the approach can tackle three types of problems crucial to a molecular understanding of a given protein: (1) it can map out 2 degrees structure, 3 degrees structure, and 4 degrees structure; (2) it can analyze conformational changes and the structural basis of regulation by covalently trapping specific conformational or signaling states; and (3) it can uncover the spatial and temporal aspects of thermal fluctuations by detecting backbone and domain dynamics. The approach can provide structural information for many proteins inaccessible to high-resolution methods. Even when a high-resolution structure is available, the approach provides complementary information about regulatory mechanisms and thermal dynamics in the native environment. Finally, the approach can be applied to an entire protein, or to a specific domain or subdomain within the full-length protein, thereby facilitating a divide-and-conquer strategy in large systems or multiprotein complexes. Rigorous application of the approach to a given protein, domain, or subdomain requires careful experimental design that adequately resolves the structural and dynamical information provided by the method. A full structural and dynamical analysis begins by scanning engineered cysteines throughout the region of interest. To determine 2 degrees structure, the solvent exposure of each cysteine is determined by measuring its chemical reactivity, and the periodicity of exposure is analyzed. To probe 3 degrees structure, 4 degrees structure, and conformational regulation, pairs of cysteines are identified that rapidly form disulfide bonds and that retain function when induced to form a disulfide bond in the folded protein or complex. Finally, to map out thermal fluctuations in a protein of known structure, disulfide formation rates are measured between distal pairs of nonperturbing surface cysteines. This chapter details these methods and illustrates applications to two proteins from the bacterial chemotaxis pathway: the periplasmic galactose binding protein and the transmembrane aspartate receptor.
The chemosensory pathway of bacterial chemotaxis has become a paradigm for
the two-component superfamily of receptor-regulated phosphorylation pathways.
This simple pathway illustrates many of the ...fundamental principles and
unanswered questions in the field of signaling biology. A molecular description
of pathway function has progressed rapidly because it is accessible to diverse
structural, biochemical, and genetic approaches. As a result, structures are
emerging for most of the pathway elements, biochemical studies are elucidating
the mechanisms of key signaling events, and genetic methods are revealing the
intermolecular interactions that transmit information between components.
Recent advances include (
a
) the first molecular picture of a
conformational transmembrane signal in a cell surface receptor, (
b
) four
new structures of kinase domains and adaptation enzymes, and (
c
)
significant new insights into the mechanisms of receptor-mediated kinase
regulation, receptor adaptation, and the phospho-activation of signaling
proteins. Overall, the chemosensory pathway and the propulsion system it
regulates provide an ideal system in which to probe molecular principles
underlying complex cellular signaling and behavior.
The aspartate receptor of the bacterial chemotaxis pathway serves as a scaffold for the formation of a multiprotein signaling complex containing the receptor and the cytoplasmic pathway components. ...Within this complex, the receptor regulates the autophosphorylation activity of histidine kinase CheA, thereby controlling the signals sent to the flagellar motor and the receptor adaptation system. The receptor cytoplasmic domain, which controls the on−off switching of CheA, possesses 14 glycine residues that are highly conserved in related receptors. In principle, these conserved glycines could be required for static turns, bends, or close packing in the cytoplasmic domain, or they could be required for conformational dynamics during receptor on−off switching. To determine which glycines are essential and to probe their functional roles, we have substituted each conserved glycine with both alanine and cysteine, and then measured the effects on receptor function in vivo and in vitro. The results reveal a subset of six glycines which are required for receptor function during cellular chemotaxis. Two of these essential glycines (G388 and G391) are located at a hairpin turn at the distal end of the folded cytoplasmic domain, where they are required for the tertiary fold of the signaling subdomain and for CheA kinase activation. Three other essential glycines (G338, G339, and G437) are located at the border between the adaptation and signaling subdomains, where they play key roles in CheA kinase activation and on−off switching. These three glycines form a ring around the four-helix bundle that comprises the receptor cytoplasmic domain, yielding a novel architectural feature termed a bundle hinge. The final essential glycine (G455) is located in the adaptation subdomain where it is required for on−off switching. Overall, the findings confirm that six of the 14 conserved cytoplasmic glycines are essential for receptor function because they enable helix turns and bends required for native receptor structure, and in some cases for switching between the on and off signaling states. An initial working model proposes that the novel bundle hinge enables the four-helix bundle to bend, perhaps during the assembly of the receptor trimer of dimers or during on−off switching. More generally, the findings predict that certain human disease states, including specific cancers, could be triggered by lock-on mutations at essential glycine positions that control the on−off switching of receptors and signaling proteins.
The number of known protein structures is growing exponentially (Berman et al., 2000), but the structural mapping of essential domain-domain and protein-protein interaction surfaces has advanced more ...slowly. It is particularly difficult to analyze the interaction surfaces of membrane proteins on a structural level, both because membrane proteins are less accessible to high-resolution structural analysis and because the membrane environment is often required for native complex formation. The Protein-Interactions-by-Cysteine-Modification (PICM) method is a generalizable, in vitro chemical scanning approach that can be applied to many protein complexes, in both membrane-bound and soluble systems. The method begins by engineering Cys residues on the surface of a protein of known structure, then a bulky probe is coupled to each Cys residue. Next, the effects of both Cys substitution and bulky probe attachment are measured on the assembly and the activity of the target complex. Bulky probe coupling at an essential docking site disrupts complex assembly and/or activity, while coupling outside the site typically has little or no effect. PICM has been successfully applied to the core signaling complex of the E. coli and S. typhimurium chemotaxis pathway, where it has mapped out essential docking surfaces on transmembrane chemoreceptor (Tar) and histidine kinase (CheA) components (Bass and Falke, 1998; Mehan et al., 2003; Miller et al., 2006). The approach shares similarities with other important scanning methods like alanine and tryptophan scanning (Cunningham and Wells, 1989; Sharp et al., 1995a), but has two unique features: (1) functional effects are determined for both small volume (Cys) and large volume (bulky probe) side chain substitutions in the same experiment, and (2) nonperturbing positions are identified at which Cys residues and bulky probes can be introduced for subsequent biochemical and biophysical studies, without significant effects on complex assembly or activity.
Breaching the Barrier Locher, Kaspar P.; Bass, Randal B.; Rees, Douglas C.
Science (American Association for the Advancement of Science),
08/2003, Letnik:
301, Številka:
5633
Journal Article
Recenzirano
The identification of more than 360 families of transporters through biochemical and genomic analyses highlights the importance of transport processes to cells. Locher et al stress the need to breach ...the major barriers to the structural determination of membrane proteins. The functions of the bacterial transporters LacY and GlpT structures are examined.
Background: Site-directed sulfhydryl chemistry and spectroscopy can be used to probe protein structure, mechanism and dynamics
in situ. The aspartate receptor of bacterial chemotaxis is ...representative of a large family of prokaryotic and eukaryotic receptors that regulate histidine kinases in two-component signaling pathways, and has become one of the best characterized transmembrane receptors. We report here the use of cysteine and disulfide scanning to probe the helix-packing architecture of the cytoplasmic domain of the aspartate receptor.
Results: A series of designed cysteine pairs have been used to detect proximities between cytoplasmic helices in the full-length, membrane-bound receptor by measurement of disulfide-bond formation rates. Upon mild oxidation, 25 disulfide bonds form rapidly between three specific pairs of helices, whereas other helix pairs yield no detectable disulfide-bond formation. Further constraints on helix packing are provided by 14 disulfide bonds that retain receptor function in an
in vitro kinase regulation assay. Of these functional disulfides, seven lock the receptor in the conformation that constitutively stimulates kinase activity (‘lock-on’), whereas the remaining seven retain normal kinase regulation. Finally, disulfide-trapping experiments in the absence of bound kinase reveal large-amplitude relative motions of adjacent helices, including helix translations and rotations of up to 19 Å and 180°, respectively.
Conclusions: The 25 rapidly formed and 14 functional disulfide bonds identify helix–helix contacts and their register in the full-length, membrane-bound receptor–kinase complex. The results reveal an extended, rather than compact, domain architecture in which the observed helix–helix interactions are best described by a four-helix bundle arrangement. A cluster of six lock-on disulfide bonds pinpoints a region of the four-helix bundle critical for kinase activation, whereas the signal-retaining disulfides indicate that signal-induced rearrangements of this region are small enough to be accommodated by disulfide-bond flexibility (≤ 1.2 Å). In the absence of bound kinase, helix packing within the cytoplasmic domain is highly dynamic.
The passage of most molecules across biological membranes is mediated by specialized integral membrane proteins known as channels and transporters. Although these transport families encompass a wide ...range of functions, molecular architectures and mechanisms, there are common elements that must be incorporated within their structures, namely the translocation pathway, ligand specificity elements and regulatory sensors to control the rate of ligand flow across the membrane. This minireview discusses aspects of the structure and mechanism of two bacterial transport systems, the stretch‐activated mechanosensitive channel of small conductance (MscS) and the ATP‐dependent vitamin B12 uptake system (BtuCD), emphasizing their general implications for transporter function.
Cysteine and disulfide scanning has been employed to probe the signaling domain, a highly conserved motif found in the cytoplasmic region of the aspartate receptor of bacterial chemotaxis and related ...members of the taxis receptor family. Previous work has characterized the N-terminal section of the signaling domain Bass, R. B., and Falke, J. J. (1998) J. Biol. Chem. 273, 25006−25014, while the present study focuses on the C-terminal section and the interactions between these two regions. Engineered cysteine residues are incorporated at positions Gly388 through Ile419 in the signaling domain, thereby generating a library of receptors each containing a single cysteine per receptor subunit. The solvent exposure of each cysteine is ascertained by chemical reactivity measurements, revealing a periodic pattern of buried hydrophobic and exposed polar residues characteristic of an amphipathic α-helix, denoted helix α8. The helix begins between positions R392 and Val401, then continues through the last residue scanned, Ile419. Activity assays carried out both in vivo and in vitro indicate that both the buried and exposed faces of this amphipathic helix are critical for proper receptor function and the buried surface is especially important for kinase downregulation. Patterns of disulfide bond formation suggest that helix α8, together with the immediately N-terminal helix α7, forms a helical hairpin that associates with a symmetric hairpin from the other subunit of the homodimer, generating an antiparallel four helix bundle containing helices α7, α7‘, α8, and α8‘. Finally, the protein-interactions-by-cysteine-modification (PICM) method suggests that the loop between helices α7 and α8 interacts with the kinase CheA and/or the coupling protein CheW, expanding the receptor surface implicated in kinase docking.
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