Protein kinase A (PKA) exists as several tissue-specific isoforms that through phosphorylation of serine and threonine residues of substrate proteins act as key regulators of a number of cellular processes. We here demonstrate that the human sperm-specific isoform of PKA named C alpha 2 is important for sperm motility and thus male fertility. Furthermore, we report on the first three-dimensional crystal structure of human apo C alpha 2 to 2.1 angstrom. Apo C alpha 2 displays an open conformation similar to the well-characterized apo structure of murine C alpha 1. The asymmetric unit contains two molecules and the core of the small lobe is rotated by almost 13 degrees in the A molecule relative to the B molecule. In addition, a salt bridge between Lys72 and Glu91 was observed for C alpha 2 in the apo-form, a conformation previously found only in dimeric or ternary complexes of C alpha 1. Human C alpha 2 and C alpha 1 share primary structure with the exception of the amino acids at the N-terminus coded for by an alternative exon 1. The N-terminal glycine of C alpha 1 is myristoylated and this aliphatic chain anchors the N-terminus to an intramolecular hydrophobic pocket. C alpha 2 cannot be myristoylated and the crystal structure revealed that the equivalent hydrophobic pocket is unoccupied and exposed. Nuclear magnetic resonance (NMR) spectroscopy further demonstrated that detergents with hydrophobic moieties of different lengths can bind deep into this uncovered pocket. Our findings indicate that C alpha 2 through the hydrophobic pocket has the ability to bind intracellular targets in the sperm cell, which may modulate protein stability, activity and/or cellular localization. (c) 2012 Elsevier Inc. All rights reserved.