G15SecL05Tpc3: Difference between revisions

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==Significance in Lyme Disease and Bind Briefing==

Lyme disease is a bacterial infectious disease of the skin, joints, nervous system and heart dominantly caused by the spirochete ''Borrelia burgdorferi'', transmitted to humans via the bite of the deer tick ''Ixodes scapularis''(Becker, 2005). The importance of antibodies in controlling such spirochaetal infections was underscored by the discovery of two particular antibodies with distinctive bactericidal properties: the monoclonal antibodies CB2 and H6831 (Connoly, Benach). When directed against the C-terminus of the outer-surface protein Osp-B, these fragments are bactericidal even in the absence of complements or phagocytes (Sadziene, 1994). The formation of the OspB-CB2 and OspB-H6831 complexes were dependent upon a single lysine residue in Osp-B, Lys 253 (GREEN LINK THAT), for binding and thus leading to lysis of the outer membrane of the spirochete; the structural changes that result culminate into molecular instability and protease susceptibility that eventually lead to said lysis, though the exact physiological consequences of these changes are not yet fully sequenced nor understood (Connoly, Benach). These antibodies demonstrate enormous selective pressure; growth of ''Borrelia burgdorferi'' spirochetes in their presence generated escape mutants that lacked the critical Lys 253 amino acid on Osp-B for antibody binding, and were thus less infectious in experimental mouse models and ''in vitro'' experimental assays (Connoly, Benach).

Lyme disease is a bacterial infectious disease of the skin, joints, nervous system and heart dominantly caused by the spirochete ''Borrelia burgdorferi'', transmitted to humans via the bite of the deer tick ''Ixodes scapularis''(Becker, 2005). The importance of antibodies in controlling such spirochaetal infections was underscored by the discovery of two particular antibodies with distinctive bactericidal properties: the monoclonal antibodies CB2 and H6831 (Connoly, Benach). When directed against the C-terminus of the outer-surface protein Osp-B, these fragments are bactericidal even in the absence of complements or phagocytes (Sadziene, 1994). The formation of the OspB-CB2 and OspB-H6831 complexes were dependent upon a single lysine residue in Osp-B, Lys 253 ('''GREEN LINK THAT'''), for binding and thus leading to lysis of the outer membrane of the spirochete; the structural changes that result culminate into molecular instability and protease susceptibility that eventually lead to said lysis, though the exact physiological consequences of these changes are not yet fully sequenced nor understood (Connoly, Benach). These antibodies demonstrate enormous selective pressure; growth of ''Borrelia burgdorferi'' spirochetes in their presence generated escape mutants that lacked the critical Lys 253 amino acid on Osp-B for antibody binding, and were thus less infectious in experimental mouse models and ''in vitro'' experimental assays (Connoly, Benach).

==Osp-B/Antibody Binding Processes==

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==H6831==

[[Image:http___blackboard.stonybrook.png|right|170px]]

Crytsallization, binding and X-ray diffraction analysis of Osp-B along with a sampling of its complement-independent antibodies have shown that its structure is analogous to that of Osp-A, with a significant difference: within each borrelia species, Osp-A is largely invariant in amino acid sequence and antigenic reactivity, but Osp-B varies significantly (Becker, 2005). These variations inhibit the ability of scientists to develop Osp-B as a protective vaccine against Lyme borreliosis. Osp-B’s variations are primarily expressed as significant differences of reactivity with monoclonal antibodies and truncation of the C-terminus; this proves problematic for vaccine development since most protective antibodies targeted against Osp-A and Osp-B are generally directed toward this terminus region. Variations such as truncation in Osp-B can inhibit this process making the vaccine ineffective, though certain complement independent antibodies such as the samples examined in the crystallization process have been found to bind and lyse Osp-B in the absence of complement.

H6831 is a specific IgG class antigen binding fragment that has a series of significant residues that bind to the epitope on Osp-B and lyse it without the aid of a complement, resulting in a unique molecular interaction that causes a physical change in the shape of Osp-B upon binding. In order to better analyze the changes, crystallized structures of Osp-B and the Osp-B H6831 complex were taken and comparatively analyzed: X-ray diffraction showed that the structure of OspB-CT (greenlink) contains twelve anti parallel beta strands and a single alpha helix (Becker, 2005). Strands 1-4 form the Osp-B’s central sheet: this is a free standing sheet that is located at the N-terminus of the protein. The remaining strands 5-12 form two additional sheets that together with the alpha helix form a barrel-like domain. Analysis of the bound complex revealed that the central beta sheet is either destroyed or removed by proteolysis upon binding (Becker, 2005). Ultimately the structural changes to Osp-B appear to result only indirectly from the addition of Fab (Becker, 2005).

Almost the entirety of this binding process is centered around distinct residues that were discovered due to comparative analysis of Osp-A and Osp-B’s crystal structures. A charged triad is formed in both structures whose united physical form presents itself as a favorable binding site. In Osp-B, this triad consists of amino-acid residues Arg-162, Glu-184, and Arg-214. These triads form a prominent cleft with density void space in between each other. This space is well suited for an all trans-structures to bind into, and such a structure was located on the crystallized Osp-B-H6831 bound complex. This complex is a combination of Osp-B-CT residues 202-296 and the Fabs’ heavy and light chains. A notable feature of the OspB-CT-H6831 interaction is the prevalence of aromatic residues contributed by the Fab: these aromatic residues are the points at which Lys-253 on Osp-B guides itself to bind with Glu-50. Specifically, Lys 253 wedges itself between a tyrosine and a tryptophan to be able reach the Glu-50 so the binding between the two residues can occur (Becker, 2005). This interaction, along with an amine hydrogen bond and ion pair with a glutamate from the Fab heavy chain, a second hydrogen bond with a main chain carbonyl from loop 1 of Osp-B-CT, and a water mediated hydrogen bond to a histidine from the Fab heavy chain constitute the three main series of interactions in the OspB-H6831 bound complex. Lys-253 is the absolute key component of the formation of this bound complex; if Lys-253 were to be substituted with another amino acid or be missing entirely via external influential mutation, the binding could not occur***. Multiple studies highlight selective pressures related to this residue: several mutated B. burgdorferi spirochetes have been shown to lack the Lys-253 residue entirely, though this form of the disease is significantly less pathogenic as a consequence.

Further studies into this protein and complement-independent antibodies are important since as of now vaccinations of Osp-B have proven unreliable due to regional variations in its form. Creation of a proper vaccine has gradually grown in interest because the current antibodies created in the human body from Osp-A have been shown to have possible correlation with arthritis, an inflammatory joint disorder. In this regard, bactericidal antibodies to Osp-B may have in vivo relevance in late stage diseases and in vaccines for the elimination of spirochetes from ticks feeding on immune individuals (Escudero, 1997).

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@@ Line 9: / Line 9: @@
 ==Significance in Lyme Disease and Bind Briefing==
-Lyme disease is a bacterial infectious disease of the skin, joints, nervous system and heart dominantly caused by the spirochete ''Borrelia burgdorferi'',  transmitted to humans via the bite of the deer tick ''Ixodes scapularis''(Becker, 2005). The importance of antibodies in controlling such spirochaetal infections was underscored by the discovery of two particular antibodies with distinctive bactericidal properties: the monoclonal antibodies CB2 and H6831 (Connoly, Benach). When directed against the C-terminus of the outer-surface protein Osp-B, these fragments are bactericidal even in the absence of complements or phagocytes (Sadziene, 1994). The formation of the OspB-CB2 and OspB-H6831 complexes were dependent upon a single lysine residue in Osp-B, Lys 253 (GREEN LINK THAT), for binding and thus leading to lysis of the outer membrane of the spirochete; the structural changes that result culminate into molecular instability and protease susceptibility that eventually lead to said lysis, though the exact physiological consequences of these changes are not yet fully sequenced nor understood (Connoly, Benach). These antibodies demonstrate enormous selective pressure; growth of ''Borrelia burgdorferi'' spirochetes in their presence generated escape mutants that lacked the critical Lys 253 amino acid on Osp-B for antibody binding, and were thus less infectious in experimental mouse models and ''in vitro'' experimental assays (Connoly, Benach).
+Lyme disease is a bacterial infectious disease of the skin, joints, nervous system and heart dominantly caused by the spirochete ''Borrelia burgdorferi'',  transmitted to humans via the bite of the deer tick ''Ixodes scapularis''(Becker, 2005). The importance of antibodies in controlling such spirochaetal infections was underscored by the discovery of two particular antibodies with distinctive bactericidal properties: the monoclonal antibodies CB2 and H6831 (Connoly, Benach). When directed against the C-terminus of the outer-surface protein Osp-B, these fragments are bactericidal even in the absence of complements or phagocytes (Sadziene, 1994). The formation of the OspB-CB2 and OspB-H6831 complexes were dependent upon a single lysine residue in Osp-B, Lys 253 ('''GREEN LINK THAT'''), for binding and thus leading to lysis of the outer membrane of the spirochete; the structural changes that result culminate into molecular instability and protease susceptibility that eventually lead to said lysis, though the exact physiological consequences of these changes are not yet fully sequenced nor understood (Connoly, Benach). These antibodies demonstrate enormous selective pressure; growth of ''Borrelia burgdorferi'' spirochetes in their presence generated escape mutants that lacked the critical Lys 253 amino acid on Osp-B for antibody binding, and were thus less infectious in experimental mouse models and ''in vitro'' experimental assays (Connoly, Benach).
 ==Osp-B/Antibody Binding Processes==
@@ Line 18: / Line 18: @@
 ==H6831==
 [[Image:http___blackboard.stonybrook.png|right|170px]]
+Crytsallization, binding and X-ray diffraction analysis of Osp-B along with a sampling of its complement-independent antibodies have shown that its structure is analogous to that of Osp-A, with a significant difference: within each borrelia species, Osp-A is largely invariant in amino acid sequence and antigenic reactivity, but Osp-B varies significantly (Becker, 2005). These variations inhibit the ability of scientists to develop Osp-B as a protective vaccine against Lyme borreliosis. Osp-B’s variations are primarily expressed as significant differences of reactivity with monoclonal antibodies and truncation of the C-terminus; this proves problematic for vaccine development since most protective antibodies targeted against Osp-A and Osp-B are generally directed toward this terminus region. Variations such as truncation in Osp-B can inhibit this process making the vaccine ineffective, though certain complement independent antibodies such as the samples examined in the crystallization process have been found to bind and lyse Osp-B in the absence of complement.
+H6831 is a specific IgG class antigen binding fragment that has a series of significant residues that bind to the epitope on Osp-B and lyse it without the aid of a complement, resulting in a unique molecular interaction that causes a physical change in the shape of Osp-B upon binding. In order to better analyze the changes, crystallized structures of Osp-B and the Osp-B H6831 complex were taken and comparatively analyzed: X-ray diffraction showed that the structure of OspB-CT (greenlink) contains twelve anti parallel beta strands and a single alpha helix (Becker, 2005). Strands 1-4 form the Osp-B’s central sheet: this is a free standing sheet that is located at the N-terminus of the protein. The remaining strands 5-12 form two additional sheets that together with the alpha helix form a barrel-like domain. Analysis of the bound complex revealed that the central beta sheet is either destroyed or removed by proteolysis upon binding (Becker, 2005). Ultimately the structural changes to Osp-B appear to result only indirectly from the addition of Fab (Becker, 2005).
+Almost the entirety of this binding process is centered around distinct residues that were discovered due to comparative analysis of Osp-A and Osp-B’s crystal structures. A charged triad is formed in both structures whose united physical form presents itself as a favorable binding site. In Osp-B, this triad consists of amino-acid residues Arg-162, Glu-184, and Arg-214. These triads form a prominent cleft with density void space in between each other. This space is well suited for an all trans-structures to bind into, and such a structure was located on the crystallized Osp-B-H6831 bound complex.  This complex is a combination of Osp-B-CT residues 202-296 and the Fabs’ heavy and light chains. A notable feature of the OspB-CT-H6831 interaction is the prevalence of aromatic residues contributed by the Fab: these aromatic residues are the points at which Lys-253 on Osp-B guides itself to bind with Glu-50. Specifically, Lys 253 wedges itself between a tyrosine and a tryptophan to be able reach the Glu-50 so the binding between the two residues can occur (Becker, 2005). This interaction, along with an amine hydrogen bond and ion pair with a glutamate from the Fab heavy chain, a second hydrogen bond with a main chain carbonyl from loop 1 of Osp-B-CT, and a water mediated hydrogen bond to a histidine from the Fab heavy chain constitute the three main series of interactions in the OspB-H6831 bound complex. Lys-253 is the absolute key component of the formation of this bound complex; if Lys-253 were to be substituted with another amino acid or be missing entirely via external influential mutation, the binding could not occur***.  Multiple studies highlight selective pressures related to this residue: several mutated B. burgdorferi spirochetes have been shown to lack the Lys-253 residue entirely, though this form of the disease is significantly less pathogenic as a consequence.
+Further studies into this protein and complement-independent antibodies are important since as of now vaccinations of Osp-B have proven unreliable due to regional variations in its form. Creation of a proper vaccine has gradually grown in interest because the current antibodies created in the human body from Osp-A have been shown to have possible correlation with arthritis, an inflammatory joint disorder. In this regard, bactericidal antibodies to Osp-B may have in vivo relevance in late stage diseases and in vaccines for the elimination of spirochetes from ticks feeding on immune individuals (Escudero, 1997).