RESUMO
L-carnosine is a bioactive dipeptide (beta-alanyl-L-histidine) present in mammalian tissues, including the central nervous system, and has potential neuroprotective and neurotransmitter functions. In mammals, two types of L-carnosine-hydrolyzing enzymes (CN1 and CN2) have been cloned thus far, and they have been classified as metallopeptidases of the M20 family. The enzymatic activity of CN2 requires Mn(2+), and CN2 is inhibited by a nonhydrolyzable substrate analog, bestatin. Here, we present the crystal structures of mouse CN2 complexed with bestatin together with Zn(2+) at a resolution of 1.7 A and that with Mn(2+) at 2.3 A CN2 is a homodimer in a noncrystallographic asymmetric unit, and the Mn(2+) and Zn(2+) complexes closely resemble each other in the overall structure. Each subunit is composed of two domains: domain A, which is complexed with bestatin and two metal ions, and domain B, which provides the major interface for dimer formation. The bestatin molecule bound to domain A interacts with several residues of domain B of the other subunit, and these interactions are likely to be essential for enzyme activity. Since the bestatin molecule is not accessible to the bulk water, substrate binding would require conformational flexibility between domains A and B. The active site structure and substrate-binding model provide a structural basis for the enzymatic activity and substrate specificity of CN2 and related enzymes.
Assuntos
Dipeptidases/química , Leucina/análogos & derivados , Metaloexopeptidases/química , Modelos Moleculares , Animais , Sítios de Ligação , Dimerização , Dipeptidases/antagonistas & inibidores , Dipeptidases/genética , Dipeptidases/metabolismo , Leucina/química , Manganês/química , Manganês/metabolismo , Metaloexopeptidases/antagonistas & inibidores , Metaloexopeptidases/genética , Metaloexopeptidases/metabolismo , Camundongos , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Especificidade por Substrato , Zinco/química , Zinco/metabolismoRESUMO
Mammalian tissues contain several histidine-containing dipeptides, of which L-carnosine is the best characterized and is found in various tissues including the brain and skeletal muscles. However, the mechanism for its biosynthesis and degradation have not yet been fully elucidated. Crystallographic study of carnosinase CN2 from mouse has been undertaken in order to understand its enzymatic mechanism from a structural viewpoint. CN2 was crystallized by the hanging-drop vapour-diffusion technique using PEG 3350 as a precipitant. Crystals were obtained in complex with either Mn(2+) or Zn(2+). Both crystals of CN2 belong to the monoclinic space group P2(1) and have almost identical unit-cell parameters (a = 54.41, b = 199.77, c = 55.49 A, beta = 118.52 degrees for the Zn(2+) complex crystals). Diffraction data were collected to 1.7 and 2.3 A for Zn(2+) and Mn(2+) complex crystals, respectively, using synchrotron radiation. Structure determination is ongoing using the multiple-wavelength anomalous diffraction (MAD) method.
Assuntos
Dipeptidases/química , Animais , Cristalização , Cristalografia por Raios X , Dipeptidases/genética , Dipeptidases/isolamento & purificação , CamundongosRESUMO
SHPS-1, a receptor-type transmembrane protein, is abundantly expressed in neural and myeloid tissues. The most amino-terminal immunoglobulin-like domain of SHPS-1 plays an important role in a variety of cell functions by binding its ligand CD47. Interaction between SHPS-1 and CD47 is thought to be involved in negative regulation of phagocytosis. The ligand-binding domain of rat SHPS-1 was purified and crystallized using the vapour-diffusion method with the solution-stirring technique. Preliminary X-ray diffraction data were collected from SHPS-1 crystals to 2.8 A resolution and reduced to primitive hexagonal space group P622. Unit-cell parameters are a = b = 100.5, c = 101.3 A.
