Lack of catalytic activity of a murine mRNA cytoplasmic serine hydroxymethyltransferase splice variant: evidence against alternative splicing as a regulatory mechanism.

Mammalian serine hydroxymethyltransferase (SHMT) is a tetrameric, pyridoxal phosphate-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single-carbon units for purine, thymidine, and methionine biosynthesis. Cytoplasmic SHMT (cSHMT) has been postulated to channel one-carbon substituted folates to various folate-dependent enzymes, and alternative splicing of the cSHMT transcript may be a mechanism that enables specific protein-protein interactions. The cytoplasmic isozyme is expressed from species-specific and tissue-specific alternatively spliced transcripts that encode proteins with modified carboxy-terminal domains, while the mitochondrial isozyme is expressed from a single transcript. While the full-length mouse and human cSHMT proteins are 91% identical, their alternatively spliced transcripts differ. The murine cSHMT gene is expressed as two transcripts. One transcript encodes a full-length 55 kDa active enzyme (cSHMT), while the other transcript encodes a 35 kDa protein (McSHMTtr). The McSHMTtr protein present in mouse liver and kidney does not bind 5-formyltetrahydrofolate, nor does it oligomerize with the full-length cSHMT enzyme. While recombinant cSHMT-glutathione S-transferase fusion proteins form tetramers and are catalytically active, McSHMTtr-glutathione S-transferase fusion proteins are catalytically inactive, do not form heterotetramers, and do not bind pyridoxal phosphate. Analysis of the murine cSHMT crystal structure indicates that the active site lysine that normally binds pyridoxal phosphate in the cSHMT protein is exposed to solvent in the McSHMTtr protein, preventing stable formation of a Schiff base with pyridoxal phosphate. Modeling studies suggest that the human cSHMT proteins expressed from alternatively spliced transcripts are inactive as well. Therefore, channeling mechanisms enabling specific protein-protein interactions of active enzymes are not based on cSHMT alternative splicing.

Structure of a murine cytoplasmic serine hydroxymethyltransferase quinonoid ternary complex: evidence for asymmetric obligate dimers.

Serine hydroxymethyltransferase (SHMT) is a pyridoxal phosphate-dependent enzyme that catalyzes the reversible conversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single carbon units for purine, thymidine, and methionine biosynthesis. The enzyme is a homotetramer comprising two obligate dimers and four pyridoxal phosphate-bound active sites. The mammalian enzyme is present in cells in both catalytically active and inactive forms. The inactive form is a ternary complex that results from the binding of glycine and 5-formyltetrahydrofolate polyglutamate, a slow tight-binding inhibitor. The crystal structure of a close analogue of the inactive form of murine cytoplasmic SHMT (cSHMT), lacking only the polyglutamate tail of the inhibitor, has been determined to 2.9 A resolution. This first structure of a ligand-bound mammalian SHMT allows identification of amino acid residues involved in substrate binding and catalysis. It also reveals that the two obligate dimers making up a tetramer are not equivalent; one can be described as "tight-binding" and the other as "loose-binding" for folate. Both active sites of the tight-binding dimer are occupied by 5-formyltetrahydrofolate (5-formylTHF), whose N5-formyl carbon is within 4 A of the glycine alpha-carbon of the glycine-pyridoxal phosphate complex; the complex appears to be primarily in its quinonoid form. In the loose-binding dimer, 5-formylTHF is present in only one of the active sites, and its N5-formyl carbon is 5 A from the glycine alpha-carbon. The pyridoxal phosphates appear to be primarily present as geminal diamine complexes, with bonds to both glycine and the active site lysine. This structure suggests that only two of the four catalytic sites on SHMT are catalytically competent and that the cSHMT-glycine-5-formylTHF ternary complex is an intermediate state analogue of the catalytic complex associated with serine and glycine interconversion.

A system for integrated collection and analysis of crystallographic diffraction data.

A set of linked software modules has been installed at the A1 station of CHESS, the Cornell High Energy Synchrotron Source, which, with the underlying hardware, allows crystallographic users of the facility to evaluate crystals, collect diffraction images and process the images rapidly and with assurance of the quality of the resultant data, thereby making most efficient use of their beam time. The system includes a CCD detector and its controlling software, with a graphical user interface, a convenient oscillation camera featuring automated alignment with the X-ray beam, a program for optimizing crystal rotation range, and the HKL data-reduction package. Principles embodied in this system are applicable to other facilities where crystallographic data are routinely collected.

The refined structure of vitamin D-dependent calcium-binding protein from bovine intestine. Molecular details, ion binding, and implications for the structure of other calcium-binding proteins.

The structure of bovine intestinal calcium-binding protein (ICaBP) has been determined crystallographically at a resolution of 2.3 A and refined by a least squares technique to an R factor of 17.8%. The refined structure includes all 600 non-hydrogen protein atoms, two bound calcium ions, and solvent consisting of one sulfate ion and 36 water molecules. The molecule consists of two helix-loop-helix calcium-binding domains known as EF hands, connected by a linker containing a single turn of helix. Helix-helix interactions are primarily hydrophobic, but also include a few strategic hydrogen bonds. Most of the hydrogen bonds, however, are found in the calcium-binding loops, where they occur both within a single loop and between the two. Examination of the hydrogen bonding patterns in the calcium-binding loops of ICaBP and the related protein, parvalbumin, reveals several conserved hydrogen bonds which are evidently important for loop stabilization. The primary and tertiary structural features which promote the formation of an EF hand were originally identified from the structure of parvalbumin. They are modified in light of the ICaBP structure and considered as they apply to other calcium-binding proteins. The C-terminal domain of ICaBP is a normal EF hand, with ion binding properties similar to those of the calmodulin hands, but the N-terminal domain is a variant hand whose calcium ligands are mostly peptide carbonyls. Relative to a normal EF hand, this domain exhibits a similar KD for calcium binding but a greatly reduced affinity for calcium analogs such as cadmium and the lanthanide series. Lanthanides in particular may be inappropriate models for calcium in this system.