Bradford A. Jameson

The surrogate light chain in B-cell development

The proteins encoded by the VpreB and lambda 5 genes associate with each other to form a light (L) chain-like structure, the surrogate L chain. It can form Ig-like complexes with three partners-the classical heavy (H) chain, the DHJHC mu-protein, or the newly discovered p55 chain; these are expressed on the surface of pre-B cells at different stages of development. Here, Fritz Melchers and colleagues review the structures of the VpreB and lambda 5 genes in mouse and their relatives in humans, describe their pattern of expression, and speculate on their possible evolution and functions.

Structure based design and characterization of peptides that inhibit IgE binding to its high-affinity receptor

We have designed synthetic peptide inhibitors of the interaction between IgE and its high affinity receptor, FcεRI. The structure of the second domain of CD2 was used as a modelling template for the second α-chain domain of FcεRI, the C-C′ loop of which has been implicated in the interaction with IgE. An L-amino acid peptide and a retro-enantiomeric D-amino acid peptide were designed to mimic the conformation of the C-C′ region. Both peptides were cyclized by disulphide bond formation between terminal cysteine residues, and show mirror image symmetry by circular dichroism analysis. The C-C′ peptide mimics act as competitive inhibitors of lgE binding. The cyclic L- and retro D-peptides exhibited K Ds of approximately 3 μM and 11 μM, respectively, for IgE. Further, the peptides inhibit IgE-mediated mast cell degranulation, an in vitro model of an allergic response.

5-HT1B receptors play a prominent role in the proliferation of T-lymphocytes

Serotonin plays a role in T cell activation, but there is no clear consensus of which of the 14 serotonergic receptors control this activations pathway. We have used a broad range of serotonergic receptor antagonists to define the functional involvement of these receptors governing the proliferation of primary T cells as well as in T cell lines. Our data shows that antagonism of the 5-HT(1B) receptor inhibits the proliferation of both human and murine primary helper T cells and of human helper T cell lines. As a whole, our data suggest that other serotonergic receptors may contribute to the proliferative signals, but the 5-HT(1B) receptor plays the most dominant role.

Identification of overlapping but distinct cAMP and cGMP interaction sites with cyclic nucleotide phosphodiesterase 3A by site-directed mutagenesis and molecular modeling based on crystalline PDE4B

Cyclic nucleotide phosphodiesterase 3A (PDE3A) hydrolyzes cAMP to AMP, but is competitively inhibited by cGMP due to a low kcat despite a tight Km. Cyclic AMP elevation is known to inhibit all pathways of platelet activation, and thus regulation of PDE3 activity is significant. Although cGMP elevation will inhibit platelet function, the major action of cGMP in platelets is to elevate cAMP by inhibiting PDE3A. To investigate the molecular details of how cGMP, a similar but not identical molecule to cAMP, behaves as an inhibitor of PDE3A, we constructed a molecular model of the catalytic domain of PDE3A based on homology to the recently determined X‐ray crystal structure of PDE4B. Based on the excellent fit of this model structure, we mutated nine amino acids in the putative catalytic cleft of PDE3A to alanine using site‐directed mutagenesis. Six of the nine mutants (Y751A, H840A, D950A, F972A, Q975A, and F1004A) significantly decreased catalytic efficiency, and had kcat/Km less than 10% of the wild‐type PDE3A using cAMP as substrate. Mutants N845A, F972A, and F1004A showed a 3‐ to 12‐fold increase of Km for cAMP. Four mutants (Y751A, H840A, D950A, and F1004A) had a 9‐ to 200‐fold increase of Ki for cGMP in comparison to the wild‐type PDE3A. Studies of these mutants and our previous study identified two groups of amino acids: E866 and F1004 contribute commonly to both cAMP and cGMP interactions while N845, E971, and F972 residues are unique for cAMP and the residues Y751, H836, H840, and D950 interact with cGMP. Therefore, our results provide biochemical evidence that cGMP interacts with the active site residues differently from cAMP.

Differential activation and inhibition of human platelet thrombin receptors by structurally distinct α-, β- and γ-thrombin

The development of drugs to neutralize the action of thrombin has to date focused on the α form of the protease. It is generally agreed that inactive prothrombin is proteolytically converted to active α-thrombin which may be further hydrolyzed to β- and γ-thrombin. While all three forms of the enzyme retain catalytic activities, only α-thrombin is presumed to be physiologically important. The β- and γ-thrombin are presumed to be degradation products of no physiological significance. Our demonstration that β- and γ-thrombin selectively activate PAR-4 in this and a previous report (J. Biol. Chem. 276, 21173–21183, 2001) necessitates a reevaluation of how we view their physiological roles and how we approach the pharmacological regulation of their actions. β-Thrombin, like γ-thrombin, at nM levels selectively activates PAR-4. This was demonstrated by full retention of aggregatory activity with platelets whose PAR-1 and GP Ib receptors were inactivated. Furthermore, the β-thrombin response was abrogated by desensitizing platelets with suboptimal levels of the thrombin receptor activating peptide for PAR-4 (TRAP-4). For β-thrombin and γ-thrombin to have a physiological role, it is necessary to show they can be generated under physiological conditions. We demonstrate, for the first time, that α-thrombin is hydrolyzed in less than 1 min by activated factor X at physiological pH, in vitro. This implies that α-thrombin may be rapidly converted to β-thrombin and/or γ-thrombin in vivo in the proper microenvironment. The differential activation of the three platelet thrombin receptors by α-, β- and γ-thrombin implies selective structural variations between these thrombin species. Structural differences are likely to account for the marked differential responses observed with the antithrombotic, hirudin, which inhibits α-thrombin, is a slightly weaker inhibitor of β- thrombin and a very weak inhibitor of γ-thrombin-induced platelet aggregations. The converse order of inhibition is observed with the physiological protease inhibitor, α1-antitrypsin. Finally, a non-traditional inhibitor, histone-1, selectively inhibits only β- and γ-thrombin, primarily at the receptor level of PAR-4 rather than on the thrombin molecule. Trypsin, like β- and γ-thrombin, activates PAR-4 and is also inactive with TRAP-4 desensitized platelets. Therefore, it was reasoned that trypsin would be more structurally similar to γ-thrombin than to α-thrombin. The analysis of the crystalline structures of α-, γ-thrombin and trypsin from the databases confirm that this is the case. These findings should help to elucidate structure-function relationships of the different thrombins and may aid in the development of new anti-thrombotic drugs.

Rational design of a peptide analog of the L3T4 CDR3-like region

The CD4 glycoprotein functions in synergy with the T cell receptor complex to generate the signals involved in T cell activation. The exact role CD4 plays in this process is not fully understood. In an attempt to further define the role of CD4 in T helper activation, we developed a structural model of the mouse CD4 (L3T4) based on the X-ray crystal structure of human CD4. From this model a synthetic peptide analog was designed to mimic the surface of the CDR3-like region of L3T4. In general, the inherent flexibility and consequent solution behavior of linear synthetic peptides have limited their application in investigating receptor-ligand interactions. We attempted to overcome these obstacles by limiting the potential peptide conformers using the rigidity of a novel turn motif and a covalent cyclization. The use of small synthetic analogs that mimic the activity of the parent protein allows the dissection of complex proteins into biologically relevant substructures. In this study we have designed an L3T4 CDR3 analog exhibiting highly specific inhibition of L3T4-dependent responses. Since the activity of the peptide maps directly to the T cell, it appears that the analog acts by uncoupling a CD4 association, and suggests a direct involvement of the CDR3-like region in cis-type interaction critical to the transduction of activation signals.

New Insights from the Structure-Function Analysis of the Catalytic Region of Human Platelet Phosphodiesterase 3A A ROLE FOR THE UNIQUE 44-AMINO ACID INSERT

Human phosphodiesterase 3A (PDE3A) degrades cAMP, the major inhibitor of platelet function, thus potentiating platelet function. Of the 11 human PDEs, only PDE3A and 3B have 44-amino acid inserts in the catalytic domain. Their function is not clear. Incubating Sp-adenosine-3′,5′-cyclic-S-(4-bromo-2,3-di-oxobutyl) monophosphorothioate (Sp-cAMPS-BDB) with PDE3A irreversibly inactivates the enzyme. High pressure liquid chromatography (HPLC) analysis of a tryptic digest yielded an octapeptide within the insert of PDE3A ((K)T806YNVTDDK813), suggesting that a substrate-binding site exists within the insert. Because Sp-cAMPS-BDB reacts with nucleophilic residues, mutants Y807A, D811A, and D812A were produced. Sp-cAMPS-BDB inactivates D811A and D812A but not Y807A. A docking model showed that Tyr807 is 3.3 angstroms from the reactive carbon, whereas Asp811 and Asp812 are >15 angstroms away from Sp-cAMPS-BDB. Y807A has an altered Km but no change in kcat. Activity of wild type but not Y807A is inhibited by an anti-insert antibody. These data suggest that Tyr807 is modified by Sp-cAMPS-BDB and involved in substrate binding. Because the homologous amino acid in PDE3B is Cys792, we prepared the mutant Y807C and found that its Km and kcat were similar to the wild type. Moreover, Sp-cAMPS-BDB irreversibly inactivates Y807C with similar kinetics to wild type, suggesting that the tyrosine may, like the cysteine, serve as a H donor. Kinetic analyses of nine additional insert mutants reveal that H782A, T810A, Y814A, and C816S exhibit an altered kcat but not Km, indicating that catalysis is modulated. We document a new functional role for the insert in which substrate binding may produce a conformational change. This change would allow the substrate to bind to Tyr807 and other amino acids in the insert to interact with residues important for catalysis in the active site cleft.

Rational design of cytotoxic T-cell inhibitors.

This study describes the use of the CD8/major histocompatibility complex (MHC) class I crystal structure as a template for the de novo design of low-molecular-weight surface mimetics. The analogs were designed from a local surface region on the CD8 α-chain directly adjacent to the bound MHC class I, to block the protein associations in the T-cell activation cluster that occur upon stimulation of the cytotoxic T lymphocytes (CTLs). One small conformationally restrained peptide showed dose-dependent inhibition of a primary allogeneic CTL assay while having no effect on the CD4-dependent mixed lymphocyte reaction (MLR). The analogs activity could be modulated through subtle changes in its side chain composition. Administration of the analog prevented CD8-dependent clearance of a murine retrovirus in BALB/c mice. In C57BL/6 mice challenged with the same retrovirus, the analog selectively inhibited the antiviral CTL responses without affecting the ability of the CTLs to generate robust allogeneic responses.

Factor XI: structure-function relationships utilizing monoclonal antibodies protein modification, computational chemistry, and rational synthetic peptide design.

Publisher Summary Coagulation factor XI is a plasma glycoprotein involved in the initiation of the intrinsic coagulation pathway. Factor XI is unique among coagulation proteins because it is a disulfide-linked homodimer with each identical monomer containing 607 amino acids. This chapter focuses on the structure–function relationships utilizing monoclonal antibodies, protein modification, computational chemistry, and rational synthetic peptide design of factor XI. Utilizing conformationally constrained synthetic peptides and sensitive and specific assays reflecting binding of factor XI (XIa) to either high-M r kininogen or factor IX, two potential heavy-chain-related surfaces utilized for binding high-M r kininogen and factor IX are available. The substrate binding site appears to consist of a sequence of amino acids from Ala–134 through Leu-172 of the A2 domain of the factor XI heavy chain that contains three stem-loop structures, which together comprise a continuous surface utilized for binding factor IX.

Understanding our link to the Great Apes—the 2003 Benjamin Franklin Medal in Life Sciences awarded to Jane Goodall

Abstract Jane Goodall is the recipient of the 2003 Benjamin Franklin Medal in Life Sciences for her long-term scientific studies of chimpanzee behavior. Not only were her studies the first of their kind, her meticulous behavioral recordings led to major changes in our understanding of the social links that exist between ourselves and our closest living relative, the chimpanzee. In discussing the importance of Dr. Goodalls work, Stephen Jay Gould (the late Alexander Agassiz Professor of Zoology at Harvard University) said ... “Science gains enormous power in replications of observations, but Homo sapiens is a single species and we can never know, by studying ourselves alone, whether important aspects of our behaviors and mental capacities reflect an ancestral evolutionary heritage (transmogrified through our uniquely evolved intelligence and its social correlates), or new features evolved or socially acquired only by our lineage. Chimpanzees are the best natural experiment we will ever have for exploring this central question, for chimps are our closest genealogical cousins and therefore hold more of our common evolutionary heritage than any other species can. Chimpanzees are not so much the shadow of man as our mirror, only slightly blurred by the mists of time”.