Zinc [Zn(II)] is essential in all Domains of life. The human pathogen Streptococcus pneumoniae must acquire Zn(II) from host tissues in order to colonise and cause disease. To achieve this, it utilises the ATP-binding cassette transporter, AdcCB, and two solute-binding proteins, AdcA and AdcAII. Although both proteins deliver Zn(II) to the AdcCB transporter, AdcAII has a greater role in overcoming host Zn(II) restriction during initial infection. Despite this, the molecular details of selective Zn(II) acquisition by AdcAII are poorly understood. To date, our understanding of the Zn(II)-binding mechanism has been based solely on the crystal structure of Zn(II)-bound AdcAII, with an open, metal-free conformation remaining refractory to crystallographic approaches. As a consequence, the conformational changes that occur within AdcAII upon Zn(II)-binding remain unknown. Here, we overcame this challenge by individually mutating each of the four Zn(II)-coordinating residues at the metal-binding site, and performing structural and biochemical analyses on the variant isoforms. Structural analyses revealed that specific regions within AdcAII underwent conformational changes due to their intimate coupling with the metal-binding residues. Quantitative in vitro metal-binding experiments, affinity determination and phenotypic analyses revealed that two of the four coordinating residues had essential contributions to the Zn(II)-binding mechanism of AdcAII. Intriguingly, only one of these residues had a direct role in structural rearrangements within AdcAII. These analyses also showed that AdcAII interacts with a broad range of first-row transition metal ions, in contrast to AdcA. Collectively, our findings provide detailed insights into the distinct mechanism of metal binding used by AdcAII to acquire essential Zn(II) ions.