Crystal structures of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed with nucleic acid: functional implications for template-primer binding to the fingers domain.

Reverse transcriptase (RT) serves as the replicative polymerase for retroviruses by using RNA and DNA-directed DNA polymerase activities coupled with a ribonuclease H activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain detailed structural information about nucleic acid interactions with reverse transcriptase, we have determined crystal structures at 2.3 A resolution of an N-terminal fragment from Moloney murine leukemia virus reverse transcriptase complexed to blunt-ended DNA in three distinct lattices. This fragment includes the fingers and palm domains from Moloney murine leukemia virus reverse transcriptase. We have also determined the crystal structure at 3.0 A resolution of the fragment complexed to DNA with a single-stranded template overhang resembling a template-primer substrate. Protein-DNA interactions, which are nearly identical in each of the three lattices, involve four conserved residues in the fingers domain, Asp114, Arg116, Asn119 and Gly191. DNA atoms involved in the interactions include the 3'-OH group from the primer strand and minor groove base atoms and sugar atoms from the n-2 and n-3 positions of the template strand, where n is the template base that would pair with an incoming nucleotide. The single-stranded template overhang adopts two different conformations in the asymmetric unit interacting with residues in the beta4-beta5 loop (beta3-beta4 in HIV-1 RT). Our fragment-DNA complexes are distinct from previously reported complexes of DNA bound to HIV-1 RT but related in the types of interactions formed between protein and DNA. In addition, the DNA in all of these complexes is bound in the same cleft of the enzyme. Through site-directed mutagenesis, we have substituted residues that are involved in binding DNA in our crystal structures and have characterized the resulting enzymes. We now propose that nucleic acid binding to the fingers domain may play a role in translocation of nucleic acid during processive DNA synthesis and suggest that our complex may represent an intermediate in this process.

Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexes.

Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragnment retain polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.

Structure of the bovine eye lens protein gammaB(gammaII)-crystallin at 1.47 A.

The molecular structure of calf gammaB-crystallin (previously called gammaII), a lens-specific protein, has been refined to a crystallographic R factor of 18.1% for all reflection data, between 8.0 and 1.47 A, 25 959 hkl measured at 293 (1) K. 230 water molecules have been defined by difference Fourier techniques and included in a restrained least-squares refinement. Difference Fourier maps clearly indicated the presence of multiple sites for the sulfur atoms of Cys 18 and Cys 22 which were therefore given coupled second-site occupancies during the refinement. The sulfur atom in the major position of Cys 22 is in the reduced state. Either of the Cys 18 sites can form a high-energy disulfide bridge with the minor position of Cys 22. The position of the carboxy terminus and many other surface side chains have been further defined including the RGD signal peptide. The hydration of the backbone and the interdomain region has been analysed. 27 water molecules make extensive contacts to a single protein molecule and thus contribute to its stability.